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1.  Oral keratinocytes support non-replicative infection and transfer of harbored HIV-1 to permissive cells 
Retrovirology  2008;5:66.
Background
Oral keratinocytes on the mucosal surface are frequently exposed to HIV-1 through contact with infected sexual partners or nursing mothers. To determine the plausibility that oral keratinocytes are primary targets of HIV-1, we tested the hypothesis that HIV-1 infects oral keratinocytes in a restricted manner.
Results
To study the fate of HIV-1, immortalized oral keratinocytes (OKF6/TERT-2; TERT-2 cells) were characterized for the fate of HIV-specific RNA and DNA. At 6 h post inoculation with X4 or R5-tropic HIV-1, HIV-1gag RNA was detected maximally within TERT-2 cells. Reverse transcriptase activity in TERT-2 cells was confirmed by VSV-G-mediated infection with HIV-NL4-3Δenv-EGFP. AZT inhibited EGFP expression in a dose-dependent manner, suggesting that viral replication can be supported if receptors are bypassed. Within 3 h post inoculation, integrated HIV-1 DNA was detected in TERT-2 cell nuclei and persisted after subculture. Multiply spliced and unspliced HIV-1 mRNAs were not detectable up to 72 h post inoculation, suggesting that HIV replication may abort and that infection is non-productive. Within 48 h post inoculation, however, virus harbored by CD4 negative TERT-2 cells trans infected co-cultured peripheral blood mononuclear cells (PBMCs) or MOLT4 cells (CD4+ CCR5+) by direct cell-to-cell transfer or by releasing low levels of infectious virions. Primary tonsil epithelial cells also trans infected HIV-1 to permissive cells in a donor-specific manner.
Conclusion
Oral keratinocytes appear, therefore, to support stable non-replicative integration, while harboring and transmitting infectious X4- or R5-tropic HIV-1 to permissive cells for up to 48 h.
doi:10.1186/1742-4690-5-66
PMCID: PMC2491655  PMID: 18637194
2.  Porphyromonas gingivalis induces CCR5-dependent transfer of infectious HIV-1 from oral keratinocytes to permissive cells 
Retrovirology  2008;5:29.
Background
Systemic infection with HIV occurs infrequently through the oral route. The frequency of occurrence may be increased by concomitant bacterial infection of the oral tissues, since co-infection and inflammation of some cell types increases HIV-1 replication. A putative periodontal pathogen, Porphyromonas gingivalis selectively up-regulates expression of the HIV-1 coreceptor CCR5 on oral keratinocytes. We, therefore, hypothesized that P. gingivalis modulates the outcome of HIV infection in oral epithelial cells.
Results
Oral and tonsil epithelial cells were pre-incubated with P. gingivalis, and inoculated with either an X4- or R5-type HIV-1. Between 6 and 48 hours post-inoculation, P. gingivalis selectively increased the infectivity of R5-tropic HIV-1 from oral and tonsil keratinocytes; infectivity of X4-tropic HIV-1 remained unchanged. Oral keratinocytes appeared to harbor infectious HIV-1, with no evidence of productive infection. HIV-1 was harbored at highest levels during the first 6 hours after HIV exposure and decreased to barely detectable levels at 48 hours. HIV did not appear to co-localize with P. gingivalis, which increased selective R5-tropic HIV-1 trans infection from keratinocytes to permissive cells. When CCR5 was selectively blocked, HIV-1 trans infection was reduced.
Conclusion
P. gingivalis up-regulation of CCR5 increases trans infection of harbored R5-tropic HIV-1 from oral keratinocytes to permissive cells. Oral infections such as periodontitis may, therefore, increase risk for oral infection and dissemination of R5-tropic HIV-1.
doi:10.1186/1742-4690-5-29
PMCID: PMC2292744  PMID: 18371227
3.  Molecular Interactions of Human Immunodeficiency Virus Type 1 with Primary Human Oral Keratinocytes 
Journal of Virology  2005;79(13):8440-8453.
Infection of the oral mucosa of human immunodeficiency virus type 1 (HIV-1)-infected individuals remains an under-evaluated and somewhat enigmatic process. Nonetheless, it is of profound importance in the ongoing AIDS pandemic, based on its potential as a site of person-to-person transmission of the virus as well as a location of HIV-1 pathogenesis and potential reservoir of disease in the setting of virally suppressive highly active antiretroviral therapy. We utilized molecular and virological techniques to analyze HIV-1 infection of primary human mucosal cells and also evaluated the proapoptotic potential of selected HIV-1 proteins in primary isolated human oral keratinocytes. Primary isolated human oral keratinocytes were plated on 0.4 μM polyethylenetetraphthalate cell culture inserts to form an in vitro oral mucosal layer. The strength of this layer in forming a barrier was determined by measuring trans-epithelial electrical current passage across the monolayer. The oral keratinocyte monolayers had trans-epithelial electrical resistance of approximately 176 to 208 Ω. For viral infectivity assays, the macrophage-tropic (R5) HIV-1 strains, YU-2 and ADA, and T-cell-line-tropic (X4), NL4-3 virions, incubated with or without deoxynucleoside triphosphates (dNTPs) and/or the polyamines spermine and spermidine, were used to infect oral keratinocytes. Of importance, polyamines and dNTPs have been shown to enhance natural endogenous reverse transcription (NERT), a step essential for early lentiviral infection, and are abundantly present in human semen. The infectivities of HIV-1 strains YU-2, ADA, and NL4-3 for these primary keratinocytes were dramatically increased by the addition of physiological concentrations of dNTPs, spermine, and spermidine. Binding and viral internalization assay studies showed no differences in these oral mucosal cells, with or without NERT-altering agents. It was also observed that the recombinant, cell-free HIV-1 proteins Nef, Tat, and gp120 (R5) induced apoptosis in primary oral keratinocytes compared with the results seen with nontreated cells or cells treated with glutathione S-transferase protein as a control under similar conditions. Microarray analyses suggested that HIV-1 gp120 and Tat induce apoptosis in primary human oral keratinocytes via the Fas/FasL apoptotic pathway, whereas induction of apoptosis by Nef occurs through both Fas/FasL and mitochondrial apoptotic pathways. Thus, these findings suggest molecular mechanisms by which semen in particular, as well as other bodily fluids such as cervicovaginal secretions, could increase oral transmission of HIV-1 via increasing infectivity in confluent and low-replicating oral keratinocytes. As well, the induction of apoptosis in human oral keratinocytes with relevant HIV-1-specific proteins suggests another potential complementary mechanism by which the oral mucosa barrier may be disrupted during HIV-1 infection in vivo.
doi:10.1128/JVI.79.13.8440-8453.2005
PMCID: PMC1143773  PMID: 15956588
4.  Oral and Vaginal Epithelial Cell Lines Bind and Transfer Cell-Free Infectious HIV-1 to Permissive Cells but Are Not Productively Infected 
PLoS ONE  2014;9(5):e98077.
The majority of HIV-1 infections worldwide are acquired via mucosal surfaces. However, unlike the vaginal mucosa, the issue of whether the oral mucosa can act as a portal of entry for HIV-1 infection remains controversial. To address potential differences with regard to the fate of HIV-1 after exposure to oral and vaginal epithelium, we utilized two epithelial cell lines representative of buccal (TR146) and pharyngeal (FaDu) sites of the oral cavity and compared them with a cell line derived from vaginal epithelium (A431) in order to determine (i) HIV-1 receptor gene and protein expression, (ii) whether HIV-1 genome integration into epithelial cells occurs, (iii) whether productive viral infection ensues, and (iv) whether infectious virus can be transferred to permissive cells. Using flow cytometry to measure captured virus by HIV-1 gp120 protein detection and western blot to detect HIV-1 p24 gag protein, we demonstrate that buccal, pharyngeal and vaginal epithelial cells capture CXCR4- and CCR5-utilising virus, probably via non-canonical receptors. Both oral and vaginal epithelial cells are able to transfer infectious virus to permissive cells either directly through cell-cell attachment or via transcytosis of HIV-1 across epithelial cells. However, HIV-1 integration, as measured by real-time PCR and presence of early gene mRNA transcripts and de novo protein production were not detected in either epithelial cell type. Importantly, both oral and vaginal epithelial cells were able to support integration and productive infection if HIV-1 entered via the endocytic pathway driven by VSV-G. Our data demonstrate that under normal conditions productive HIV-1 infection of epithelial cells leading to progeny virion production is unlikely, but that epithelial cells can act as mediators of systemic viral dissemination through attachment and transfer of HIV-1 to permissive cells.
doi:10.1371/journal.pone.0098077
PMCID: PMC4032250  PMID: 24857971
5.  PPARγ and LXR Signaling Inhibit Dendritic Cell-Mediated HIV-1 Capture and trans-Infection 
PLoS Pathogens  2010;6(7):e1000981.
Dendritic cells (DCs) contribute to human immunodeficiency virus type 1 (HIV-1) transmission and dissemination by capturing and transporting infectious virus from the mucosa to draining lymph nodes, and transferring these virus particles to CD4+ T cells with high efficiency. Toll-like receptor (TLR)-induced maturation of DCs enhances their ability to mediate trans-infection of T cells and their ability to migrate from the site of infection. Because TLR-induced maturation can be inhibited by nuclear receptor (NR) signaling, we hypothesized that ligand-activated NRs could repress DC-mediated HIV-1 transmission and dissemination. Here, we show that ligands for peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor (LXR) prevented proinflammatory cytokine production by DCs and inhibited DC migration in response to the chemokine CCL21 by preventing the TLR-induced upregulation of CCR7. Importantly, PPARγ and LXR signaling inhibited both immature and mature DC-mediated trans-infection by preventing the capture of HIV-1 by DCs independent of the viral envelope glycoprotein. PPARγ and LXR signaling induced cholesterol efflux from DCs and led to a decrease in DC-associated cholesterol, which has previously been shown to be required for DC capture of HIV-1. Finally, both cholesterol repletion and the targeted knockdown of the cholesterol transport protein ATP-binding cassette A1 (ABCA1) restored the ability of NR ligand treated cells to capture HIV-1 and transfer it to T cells. Our results suggest that PPARγ and LXR signaling up-regulate ABCA1-mediated cholesterol efflux from DCs and that this accounts for the decreased ability of DCs to capture HIV-1. The ability of NR ligands to repress DC mediated trans-infection, inflammation, and DC migration underscores their potential therapeutic value in inhibiting HIV-1 mucosal transmission.
Author Summary
Heterosexual transmission is the primary mode of HIV transmission worldwide. In the absence of an effective vaccine, there is an increasing demand for the development of effective microbicides that block HIV sexual transmission. Dendritic cells (DCs) play a critical role in HIV transmission by efficiently binding virus particles, migrating to lymph nodes, and transmitting them to CD4+ T cells, a process called trans-infection. In addition, DCs secrete proinflammatory cytokines that create a favorable environment for virus replication. DC maturation by pathogen-encoded TLR ligands or proinflammatory cytokines dramatically increases their capacity to capture HIV, migrate to lymphoid tissue, and trans-infect T cells. Here, we report that signaling through the nuclear receptors PPARγ and LXR prevents DC maturation and proinflammatory cytokine production, as well as migration. In addition, PPARγ and LXR signaling prevents efficient DC capture and transfer of infectious HIV by increasing ABCA1-mediated cholesterol efflux. Our studies suggest that PPARγ and LXR may be targets for drugs that can inhibit specific aspects of HIV mucosal transmission, namely inflammation, migration, and virus capture and transfer. These findings provide a rationale for considering PPARγ and LXR agonists as potential combination therapies with conventional anti-viral microbicides that target other aspects of mucosal HIV transmission.
doi:10.1371/journal.ppat.1000981
PMCID: PMC2895661  PMID: 20617179
6.  Stromal Down-Regulation of Macrophage CD4/CCR5 Expression and NF-κB Activation Mediates HIV-1 Non-Permissiveness in Intestinal Macrophages 
PLoS Pathogens  2011;7(5):e1002060.
Tissue macrophages are derived exclusively from blood monocytes, which as monocyte-derived macrophages support HIV-1 replication. However, among human tissue macrophages only intestinal macrophages are non-permissive to HIV-1, suggesting that the unique microenvironment in human intestinal mucosa renders lamina propria macrophages non-permissive to HIV-1. We investigated this hypothesis using blood monocytes and intestinal extracellular matrix (stroma)-conditioned media (S-CM) to model the exposure of newly recruited monocytes and resident macrophages to lamina propria stroma, where the cells take up residence in the intestinal mucosa. Exposure of monocytes to S-CM blocked up-regulation of CD4 and CCR5 expression during monocyte differentiation into macrophages and inhibited productive HIV-1 infection in differentiated macrophages. Importantly, exposure of monocyte-derived macrophages simultaneously to S-CM and HIV-1 also inhibited viral replication, and sorted CD4+ intestinal macrophages, a proportion of which expressed CCR5+, did not support HIV-1 replication, indicating that the non-permissiveness to HIV-1 was not due to reduced receptor expression alone. Consistent with this conclusion, S-CM also potently inhibited replication of HIV-1 pseudotyped with vesicular stomatitis virus glycoprotein, which provides CD4/CCR5-independent entry. Neutralization of TGF-β in S-CM and recombinant TGF-β studies showed that stromal TGF-β inhibited macrophage nuclear translocation of NF-κB and HIV-1 replication. Thus, the profound inability of intestinal macrophages to support productive HIV-1 infection is likely the consequence of microenvironmental down-regulation of macrophage HIV-1 receptor/coreceptor expression and NF-κB activation.
Author Summary
Human intestinal macrophages, unlike lymphoid tissue macrophages, brain microglia and genital (vaginal) macrophages, are profoundly incapable of supporting productive HIV-1 infection. Intriguingly, all macrophages are derived exclusively from blood monocytes, which are HIV-1 permissive after differentiation into monocyte-derived macrophages. Therefore, the unique non-permissiveness of intestinal macrophages to HIV-1 must be conferred by the intestinal mucosal microenvironment. Here we report that intestinal stroma potently blocked up-regulation of HIV-1 receptor/coreceptor CD4 and CCR5 expression during monocyte differentiation into macrophages and macrophage nuclear translocation of NF-κB, which is a critical requirement for HIV-1 transcription. These two mechanisms work collaboratively to render intestinal macrophages non-permissive to HIV-1. Harnessing this natural antiviral defense may provide a novel strategy to exploit for the prevention of infection in HIV-1 permissive cells.
doi:10.1371/journal.ppat.1002060
PMCID: PMC3102716  PMID: 21637819
7.  HIV-1 envelope, integrins and co-receptor use in mucosal transmission of HIV 
Journal of Translational Medicine  2011;9(Suppl 1):S2.
It is well established that HIV-1 infection typically involves an interaction between the viral envelope protein gp120/41 and the CD4 molecule followed by a second interaction with a chemokine receptor, usually CCR5 or CXCR4. In the early stages of an HIV-1 infection CCR5 using viruses (R5 viruses) predominate. In some viral subtypes there is a propensity to switch to CXCR4 usage (X4 viruses). The receptor switch occurs in ~ 40% of the infected individuals and is associated with faster disease progression. This holds for subtypes B and D, but occurs less frequently in subtypes A and C. There are several hypotheses to explain the preferential transmission of R5 viruses and the mechanisms that lead to switching of co-receptor usage; however, there is no definitive explanation for either. One important consideration regarding transmission is that signaling by R5 gp120 may facilitate transmission of R5 viruses by inducing a permissive environment for HIV replication. In the case of sexual transmission, infection by HIV requires the virus to breach the mucosal barrier to gain access to the immune cell targets that it infects; however, the immediate events that follow HIV exposure at genital mucosal sites are not well understood. Upon transmission, the HIV quasispecies that is replicating in an infected donor contracts through a “genetic bottleneck”, and often infection results from a single infectious event. Many details surrounding this initial infection remain unresolved. In mucosal tissues, CD4+ T cells express high levels of CCR5, and a subset of these CD4+/CCR5high cells express the integrin α4β7, the gut homing receptor. CD4+/CCR5high/ α4β7high T cells are highly susceptible to infection by HIV-1 and are ideal targets for an efficient productive infection at the point of transmission. In this context we have demonstrated that the HIV-1 envelope protein gp120 binds to α4β7 on CD4+ T cells. On CD4+/CCR5high/ α4β7high T cells, α4β7 is closely associated with CD4 and CCR5. Furthermore, α4β7 is ~3 times the size of CD4 on the cell surface, that makes it a prominent receptor for an efficient virus capture. gp120-α4β7 interactions mediate the activation of the adhesion-associated integrin LFA-1. LFA-1 facilitates the formation of virological synapses and cell-to-cell spread of HIV-1. gp120 binding to α4β7 is mediated by a tripeptide located in the V1/V2 domain of gp120. Of note, the V1/V2 domain of gp120 has been linked to variations in transmission fitness among viral isolates raising the intriguing possibility that gp120-α4β7 interactions may be linked to transmission fitness. Although many details remain unresolved, we hypothesize that gp120-α4β7 interactions play an important role in the very early events following sexual transmission of HIV and may have important implication in the design of vaccine strategies for the prevention of acquisition of HIV infection
doi:10.1186/1479-5876-9-S1-S2
PMCID: PMC3105502  PMID: 21284901
8.  Supervised and Unsupervised Self-Testing for HIV in High- and Low-Risk Populations: A Systematic Review 
PLoS Medicine  2013;10(4):e1001414.
By systematically reviewing the literature, Nitika Pant Pai and colleagues assess the evidence base for HIV self tests both with and without supervision.
Background
Stigma, discrimination, lack of privacy, and long waiting times partly explain why six out of ten individuals living with HIV do not access facility-based testing. By circumventing these barriers, self-testing offers potential for more people to know their sero-status. Recent approval of an in-home HIV self test in the US has sparked self-testing initiatives, yet data on acceptability, feasibility, and linkages to care are limited. We systematically reviewed evidence on supervised (self-testing and counselling aided by a health care professional) and unsupervised (performed by self-tester with access to phone/internet counselling) self-testing strategies.
Methods and Findings
Seven databases (Medline [via PubMed], Biosis, PsycINFO, Cinahl, African Medicus, LILACS, and EMBASE) and conference abstracts of six major HIV/sexually transmitted infections conferences were searched from 1st January 2000–30th October 2012. 1,221 citations were identified and 21 studies included for review. Seven studies evaluated an unsupervised strategy and 14 evaluated a supervised strategy. For both strategies, data on acceptability (range: 74%–96%), preference (range: 61%–91%), and partner self-testing (range: 80%–97%) were high. A high specificity (range: 99.8%–100%) was observed for both strategies, while a lower sensitivity was reported in the unsupervised (range: 92.9%–100%; one study) versus supervised (range: 97.4%–97.9%; three studies) strategy. Regarding feasibility of linkage to counselling and care, 96% (n = 102/106) of individuals testing positive for HIV stated they would seek post-test counselling (unsupervised strategy, one study). No extreme adverse events were noted. The majority of data (n = 11,019/12,402 individuals, 89%) were from high-income settings and 71% (n = 15/21) of studies were cross-sectional in design, thus limiting our analysis.
Conclusions
Both supervised and unsupervised testing strategies were highly acceptable, preferred, and more likely to result in partner self-testing. However, no studies evaluated post-test linkage with counselling and treatment outcomes and reporting quality was poor. Thus, controlled trials of high quality from diverse settings are warranted to confirm and extend these findings.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
About 34 million people (most living in resource-limited countries) are currently infected with HIV, the virus that causes AIDS, and about 2.5 million people become infected with HIV every year. HIV is usually transmitted through unprotected sex with an infected partner. HIV infection is usually diagnosed by looking for antibodies to HIV in blood or saliva. Early during infection, the immune system responds to HIV by beginning to make antibodies that recognize the virus and target it for destruction. “Seroconversion”—the presence of detectable amounts of antibody in the blood or saliva—usually takes 6–12 weeks. Rapid antibody-based tests, which do not require laboratory facilities, can provide a preliminary result about an individual's HIV status from a simple oral swab or finger stick sample within 20 minutes. However preliminary rapid positive results have to be confirmed in a laboratory, which may take a few days or weeks. If positive, HIV infection can be controlled but not cured by taking a daily cocktail of powerful antiretroviral drugs throughout life.
Why Was This Study Done?
To reduce the spread of HIV, it is essential that HIV-positive individuals get tested, change behaviors avoid transmitting the virus to other people by, for example, always using a condom during sex, and if positive get on to treatment that is available worldwide. Treatment also reduces transmission of virus to the partner and controls the virus in the community. However, only half the people currently living with HIV know their HIV status, a state of affairs that increases the possibility of further HIV transmission to their partners and children. HIV positive individuals are diagnosed late with advanced HIV infection that costs health care services. Although health care facility-based HIV testing has been available for decades, people worry about stigma, visibility, and social discrimination. They also dislike the lack of privacy and do not like having to wait for their test results. Self-testing (i.e., self-test conduct and interpretation) might alleviate some of these barriers to testing by allowing individuals to determine their HIV status in the privacy of their home and could, therefore, increase the number of individuals aware of their HIV status. This could possibly reduce transmission and, through seeking linkages to care, bring HIV under control in communities. In some communities and countries, stigma of HIV prevents people from taking action about their HIV status. Indeed, an oral (saliva-based) HIV self-test kit is now available in the US. But how acceptable, feasible, and accurate is self-testing by lay people, and will people who find themselves self-test positive seek counseling and treatment? In this systematic review (a study that uses pre-defined criteria to identify all the research on a given topic), the researchers examine these issues by analyzing data from studies that have evaluated supervised self-testing (self-testing and counseling aided by a health-care professional) and unsupervised self-testing (self-testing performed without any help but with counseling available by phone or internet).
What Did the Researchers Do and Find?
The researchers identified 21 eligible studies, two-thirds of which evaluated oral self-testing and a third of which evaluated blood-based self-testing. Seven studies evaluated an unsupervised self-testing strategy and 14 evaluated a supervised strategy. Most of the data (89%) came from studies undertaken in high-income settings. The study populations varied from those at high risk of HIV infection to low-risk general populations. Across the studies, acceptability (defined as the number of people who actually self-tested divided by the number who consented to self-test) ranged from 74% to 96%. With both strategies, the specificity of self-testing (the chance of an HIV-negative person receiving a negative test result is true negative) was high but the sensitivity of self-testing (the chance of an HIV-positive person receiving a positive test result is indeed a true positive) was higher for supervised than for unsupervised testing. The researchers also found evidence that people preferred self-testing to facility-based testing and oral self-testing to blood-based self testing and, in one study, 96% of participants who self-tested positive sought post-testing counseling.
What Do These Findings Mean?
These findings provide new but limited information about the feasibility, acceptability, and accuracy of HIV self-testing. They suggest that it is feasible to implement both supervised and unsupervised self-testing, that both strategies are preferred to facility-based testing, but that the accuracy of self-testing is variable. However, most of the evidence considered by the researchers came from high-income countries and from observational studies of varying quality, and data on whether people self-testing positive sought post-testing counseling (linkage to care) were only available from one evaluation of unsupervised self-testing in the US. Consequently, although these findings suggest that self-testing could engage individuals in finding our their HIV status and thereby help modify behavior thus, reduce HIV transmission in the community, by increasing the proportion of people living with HIV who know their HIV status. The researchers suggested that more data from diverse settings and preferably from controlled randomized trials must be collected before any initiatives for global scale-up of self-testing for HIV infection are implemented.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001414.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
NAM/aidsmap provides basic information about HIV/AIDS and summaries of recent research findings on HIV care and treatment
Information is available from Avert, an international AIDS charity on many aspects of HIV/AIDS, including information on HIV testing, and on HIV transmission and testing (in English and Spanish)
The UK National Health Service Choices website provides information about all aspects of HIV and AIDS; a “behind the headlines” article provides details about the 2012 US approval for an over-the-counter HIV home-use test
The 2012 World AIDS Day Report provides information about the percentage of people living with HIV who are aware of their HIV status in various African countries, as well as up-to-date information about the AIDS epidemic
Patient stories about living with HIV/AIDS are available through Avert; the nonprofit website Healthtalkonline also provides personal stories about living with HIV, including stories about getting a diagnosis
doi:10.1371/journal.pmed.1001414
PMCID: PMC3614510  PMID: 23565066
9.  Usage of the coreceptors CCR-5, CCR-3, and CXCR-4 by primary and cell line-adapted human immunodeficiency virus type 2. 
Journal of Virology  1997;71(11):8237-8244.
The chemokine receptors CCR-5 and CXCR-4, and possibly CCR-3, are the principal human immunodeficiency virus type 1 (HIV-1) coreceptors, apparently interacting with HIV-1 envelope, in association with CD4. Cell lines coexpressing CD4 and these chemokine receptors were infected with a panel of seven primary HIV-2 isolates passaged in peripheral blood mononuclear cells (PBMC) and three laboratory HIV-2 strains passaged in T-cell lines. The CCR-5, CCR-3, and CXCR-4 coreceptors could all be used by HIV-2. The ability to use CXCR-4 represents a major difference between HIV-2 and the closely related simian immunodeficiency viruses. Most HIV-2 strains using CCR-5 could also use CCR-3, sometimes with similar efficiencies. As observed for HIV-1, the usage of CCR-5 or CCR-3 was observed principally for HIV-2 strains derived from asymptomatic individuals, while HIV-2 strains derived from AIDS patients used CXCR-4. However, there were several exceptions, and the patterns of coreceptor usage seemed more complex for HIV-2 than for HIV-1. The two T-tropic HIV-2 strains tested used CXCR-4 and not CCR-5, while T-tropic HIV-1 can generally use both. Moreover, among five primary HIV-2 strains all unable to use CXCR-4, three could replicate in CCR-5-negative PBMC, which has not been reported for HIV-1. These observations suggest that the CCR-5 coreceptor is less important for HIV-2 than for HIV-1 and indicate that HIV-2 can use other cell entry pathways and probably other coreceptors. One HIV-2 isolate replicating in normal or CCR-5-negative PBMC failed to infect CXCR-4+ cells or the U87MG-CD4 and sMAGI cell lines, which are permissive to infection by HIV-2 but not by HIV-1. This suggests the existence of several HIV-2-specific coreceptors, which are differentially expressed in cell lines and PBMC.
PMCID: PMC192281  PMID: 9343175
10.  Infection of Macrophages and Dendritic Cells with Primary R5-Tropic Human Immunodeficiency Virus Type 1 Inhibited by Natural Polyreactive Anti-CCR5 Antibodies Purified from Cervicovaginal Secretions▿  
Heterosexual contact is the primary mode of human immunodeficiency virus (HIV) type 1 (HIV-1) transmission worldwide. The chemokine receptor CCR5 is the major coreceptor that is associated with the mucosal transmission of R5-tropic HIV-1 during sexual intercourse. The CCR5 molecule is thus a target for antibody-based therapeutic strategies aimed at blocking HIV-1 entry into cells. We have previously demonstrated that polyreactive natural antibodies (NAbs) from therapeutic preparations of immunoglobulin G and from human breast milk contain NAbs directed against CCR5. Such antibodies inhibit the infection of human macrophages and T lymphocytes by R5-tropic isolates of HIV in vitro. In the present study, we demonstrate that human immunoglobulins from the cervicovaginal secretions of HIV-seronegative or HIV-seropositive women contain NAbs directed against the HIV-1 coreceptor CCR5. Natural affinity-purified anti-CCR5 antibodies bound to CCR5 expressed on macrophages and dendritic cells and further inhibited the infection of macrophages and dendritic cells with primary and laboratory-adapted R5-tropic HIV but not with X4-tropic HIV. Natural anti-CCR5 antibodies moderately inhibited R5-tropic HIV transfer from monocyte-derived dendritic cells to autologous T cells. Our results suggest that mucosal anti-CCR5 antibodies from healthy immunocompetent donors may hamper the penetration of HIV and may be suitable for use in the development of novel passive immunotherapy regimens in specific clinical settings of HIV infection.
doi:10.1128/CVI.00463-07
PMCID: PMC2394833  PMID: 18353923
11.  Short Communication: HIV Type 1 Escapes Inactivation by Saliva via Rapid Escape into Oral Epithelial Cells 
AIDS Research and Human Retroviruses  2012;28(12):1574-1578.
Abstract
Saliva contains anti-HIV-1 factors, which show unclear efficacy in thwarting mucosal infection. When incubated in fresh, unfractionated whole saliva, infectious HIV-1 IIIb and BaL (X4- and R5-tropic, respectively) persisted from 4 to at least 30 min in a saliva concentration-dependent manner. In salivary supernatant for up to 6 h, both infectious HIV-1 strains “escaped” into immortalized oral epithelial cells; infectious BaL showed selectively enhanced escape in the presence of saliva. Fluorescently labeled HIV-1 virus-like particles entered oral epithelial cells within minutes of exposure. Using a previously unrecognized mechanism, therefore, strains of HIV-1 escape inactivation by saliva via rapid uptake into oral epithelial cells.
doi:10.1089/aid.2011.0069
PMCID: PMC3505059  PMID: 22077822
12.  Human Immunodeficiency Virus Type 1 Infection and Replication in Normal Human Oral Keratinocytes 
Journal of Virology  2003;77(6):3470-3476.
Recent epidemiologic studies show increasing human immunodeficiency virus type 1 (HIV-1) transmission through oral-genital contact. This paper examines the possibility that normal human oral keratinocytes (NHOKs) might be directly infected by HIV or might convey infectious HIV virions to adjacent leukocytes. PCR analysis of proviral DNA constructs showed that NHOKs can be infected by CXCR4-tropic (NL4-3 and ELI) and dualtropic (89.6) strains of HIV-1 to generate a weak but productive infection. CCR5-tropic strain Ba-L sustained minimal viral replication. Antibody inhibition studies showed that infection by CXCR4-tropic viral strains is mediated by the galactosylceramide receptor and the CXCR4 chemokine coreceptor. Coculture studies showed that infectious HIV-1 virions can also be conveyed from NHOKs to activated peripheral blood lymphocytes, suggesting a potential role of oral epithelial cells in the transmission of HIV infection.
doi:10.1128/JVI.77.6.3470-3476.2003
PMCID: PMC149546  PMID: 12610122
13.  Cleavage of protease-activated receptors on an immortalized oral epithelial cell line by Porphyromonas gingivalis gingipains 
Microbiology  2009;155(Pt 10):3238-3246.
Porphyromonas gingivalis activates protease-activated receptors (PARs) on oral keratinocytes, resulting in downstream signalling for an innate immune response. Activation depends on P. gingivalis gingipains, but could be confounded by lipopolysaccharide signalling through Toll-like receptors. We therefore hypothesized that P. gingivalis cleaves oral keratinocyte PARs in an Arg- (Rgp) or Lys- (Kgp) gingipain-specific manner to upregulate pro-inflammatory cytokines. Immortalized human oral keratinocytes (TERT-2) were incubated with wild-type P. gingivalis (ATCC 33277) or strains from a panel of isogenic gingipain deletion mutants: Kgp-deficient (KDP 129); Rgp-deficient (KDP 133); or Kgp- and Rgp-deficient (KDP 136). After incubation with P. gingivalis, keratinocytes were probed with specific antibodies against the N-terminus of PAR-1 and PAR-2. Using flow cytometry and immunofluorescence, receptor cleavage was marked by loss of specific antibody binding to the respective PARs. TERT-2 cells constitutively expressed high levels of PAR-1 and PAR-2, and lower levels of PAR-3. P. gingivalis ATCC 33277 cleaved PAR-1 and PAR-2 in a dose-dependent manner, while the receptors were unaffected by the protease-negative double mutant (KDP 136) at all m.o.i. tested. The single Kgp-negative mutant preferentially cleaved PAR-1, whereas the Rgp-negative mutant cleaved PAR-2. Wild-type or Kgp-negative mutant cleavage of PAR-1 upregulated expression of IL-1α, IL-1β, IL-6 and TNF-α; the Rgp-negative mutant did not modulate these cytokines. Selective cleavage of PAR-1 on oral epithelial cells by P. gingivalis Rgp therefore upregulates expression of pro-inflammatory cytokines.
doi:10.1099/mic.0.029132-0
PMCID: PMC2889418  PMID: 19608609
14.  Stress-Mediated Increases in Systemic and Local Epinephrine Impair Skin Wound Healing: Potential New Indication for Beta Blockers 
PLoS Medicine  2009;6(1):e1000012.
Background
Stress, both acute and chronic, can impair cutaneous wound repair, which has previously been mechanistically ascribed to stress-induced elevations of cortisol. Here we aimed to examine an alternate explanation that the stress-induced hormone epinephrine directly impairs keratinocyte motility and wound re-epithelialization. Burn wounds are examined as a prototype of a high-stress, high-epinephrine, wound environment. Because keratinocytes express the β2-adrenergic receptor (β2AR), another study objective was to determine whether β2AR antagonists could block epinephrine effects on healing and improve wound repair.
Methods and Findings
Migratory rates of normal human keratinocytes exposed to physiologically relevant levels of epinephrine were measured. To determine the role of the receptor, keratinocytes derived from animals in which the β2AR had been genetically deleted were similarly examined. The rate of healing of burn wounds generated in excised human skin in high and low epinephrine environments was measured. We utilized an in vivo burn wound model in animals with implanted pumps to deliver β2AR active drugs to study how these alter healing in vivo. Immunocytochemistry and immunoblotting were used to examine the up-regulation of catecholamine synthetic enzymes in burned tissue, and immunoassay for epinephrine determined the levels of this catecholamine in affected tissue and in the circulation. When epinephrine levels in the culture medium are elevated to the range found in burn-stressed animals, the migratory rate of both cultured human and murine keratinocytes is impaired (reduced by 76%, 95% confidence interval [CI] 56%–95% in humans, p < 0.001, and by 36%, 95% CI 24%–49% in mice, p = 0.001), and wound re-epithelialization in explanted burned human skin is delayed (by 23%, 95% CI 10%–36%, p = 0.001), as compared to cells or tissues incubated in medium without added epinephrine. This impairment is reversed by β2AR antagonists, is absent in murine keratinocytes that are genetically depleted of the β2AR, and is reproduced by incubation of keratinocytes with other β2AR-specific agonists. Activation of the β2AR in cultured keratinocytes signals the down-regulation of the AKT pathway, accompanied by a stabilization of the actin cytoskeleton and an increase in focal adhesion formation, resulting in a nonmigratory phenotype. Burn wound injury in excised human skin also rapidly up-regulates the intra-epithelial expression of the epinephrine synthesizing enzyme phenylethanolamine-N-methyltransferase, and tissue levels of epinephrine rise dramatically (15-fold) in the burn wounded tissue (values of epinephrine expressed as pg/ug protein ± standard error of the mean: unburned control, 0.6 ± 0.36; immediately postburn, 9.6 ± 1.58; 2 h postburn, 3.1 ± 1.08; 24 h post-burn, 6.7 ± 0.94). Finally, using an animal burn wound model (20% body surface in mice), we found that systemic treatment with βAR antagonists results in a significant increase (44%, 95% CI 27%–61%, p < 0.00000001) in the rate of burn wound re-epithelialization.
Conclusions
This work demonstrates an alternate pathway by which stress can impair healing: by stress-induced elevation of epinephrine levels resulting in activation of the keratinocyte β2AR and the impairment of cell motility and wound re-epithelialization. Furthermore, since the burn wound locally generates epinephrine in response to wounding, epinephrine levels are locally, as well as systemically, elevated, and wound healing is impacted by these dual mechanisms. Treatment with beta adrenergic antagonists significantly improves the rate of burn wound re-epithelialization. This work suggests that specific β2AR antagonists may be apt, near-term translational therapeutic targets for enhancing burn wound healing, and may provide a novel, low-cost, safe approach to improving skin wound repair in the stressed individual.
Rivkah Isseroff and colleagues describe how stress-induced elevation of epinephrine levels can impair the healing of burns in mice and suggest that β2 adrenergic receptor antagonists may have a role in improving skin wound repair.
Editors' Summary
Background.
Skin—the largest organ in the human body—protects the rest of the body against infection by forming an impervious layer over the whole external body surface. Consequently, if this layer is damaged by rubbing, cutting, or burning, it must be quickly and efficiently repaired. Wound repair (healing) involves several different processes. First, the clotting cascade stops bleeding at the wound site and immune system cells attracted into the site remove any bacteria or debris in the wound. Various factors are released by the immune cells and the other cells in and near the damaged area that encourage the migration of several different sorts of cells into the wound. These cells proliferate and prepare the wound for “re-epithelialization.” In this process, keratinocytes (a type of epithelial cell that makes a tough, insoluble protein called keratin; epithelial cells cover all the surfaces of the body) migrate into the wound site and form a new, intact epithelial layer. If any of these processes fail, the result can be a chronic (long-lasting) nonhealing wound. In particular, if the wound does not re-epithelialize, it remains open and susceptible to infection and loss of body fluids.
Why Was This Study Done?
One factor that impairs the repair of skin wounds is stress. In stressful situations (including situations in which wounds are likely to occur), the human body releases several chemicals that prepare the body for “fight or flight,” including cortisol and epinephrine (also called adrenaline). Most scientists ascribe the effects of stress on wound healing to stress-induced increases in cortisol, but might stress-induced epinephrine also affect wound healing? In this study, the researchers test whether epinephrine impairs keratinocyte migration and re-epithelialization of burn wounds (keratinocytes have a receptor for epinephrine called the β2 adrenergic receptor [β2AR] on their cell surface that allows them to respond to epinephrine). They chose to study burn wounds for two reasons. First, major burns cause a massive release of stress chemicals into the bloodstream that raises blood levels (systemic levels) of cortisol and epinephrine for days or weeks after the initial trauma. Second, despite recent therapeutic advances, many people still die from major burns (4,000 every year in the USA alone) so there is a pressing need for better ways to treat this type of wound.
What Did the Researchers Do and Find?
The researchers investigated the effects of epinephrine on wound healing in three types of experiments. First, they looked at the effect of epinephrine on keratinocytes growing in dishes (in vitro experiments). Levels of epinephrine similar to those in the blood of stressed individuals greatly inhibited the motility and migration of human keratinocytes (isolated from the foreskin of newborn babies) and of mouse keratinocytes. It also inhibited the repair of scratch wounds made in monolayers of keratinocytes growing on dishes. Treatment of the cultures with a β2AR antagonist (a chemical that prevents epinephrine activating the β2AR) reversed the effects of epinephrine. In addition, the migration of mouse keratinocytes that had been genetically altered so that they did not express β2AR was not inhibited by epinephrine. Next, the researchers investigated the healing of burn wounds made in small pieces of human skin growing in dishes (ex vivo experiments). Burn injuries rapidly increased the amount of epinephrine in these tissue explants, they report, and treatment of the explants with a βAR antagonist (an inhibitor of all types of βARs) greatly increased wound re-epithelialization. Finally, the researchers report that the re-epithelialization of burn wounds in living mice was improved when the mice were treated with a β2AR antagonist.
What Do These Findings Mean?
These findings reveal a second pathway by which stress can impair wound healing. They show that stress-induced increases in systemic and local epinephrine activate β2ARs on keratinocytes and that this activation inhibits keratinocyte motility and wound re-epithelialization. Although results obtained in animals do not always reflect what happens in people, the finding that the treatment of mice with β2AR antagonists improves the rate of burn wound re-epithelialization, suggests that beta blockers—drugs that inhibit all βARs and that are widely used to treat high blood pressure and to prevent heart disease—or specific β2AR antagonists might provide a new therapeutic approach to the treatment of burns and, perhaps, chronic nonhealing wounds.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000012.
Wikipedia has pages on wound healing, burn injuries, and epinephrine (Note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The MedlinePlus Encyclopedia has a page on burns (in English and Spanish)
MedlinePlus provides links to other information on burns (in English and Spanish)
doi:10.1371/journal.pmed.1000012
PMCID: PMC2621262  PMID: 19143471
15.  HIV Traffics through a Specialized, Surface-Accessible Intracellular Compartment during trans-Infection of T Cells by Mature Dendritic Cells 
PLoS Pathogens  2008;4(8):e1000134.
In vitro, dendritic cells (DCs) bind and transfer intact, infectious HIV to CD4 T cells without first becoming infected, a process known as trans-infection. trans-infection is accomplished by recruitment of HIV and its receptors to the site of DC–T cell contact and transfer of virions at a structure known as the infectious synapse. In this study, we used fluorescent microscopy to track individual HIV particles trafficking in DCs during virus uptake and trans-infection. Mature DCs rapidly concentrated HIV into an apparently intracellular compartment that lacked markers characteristic of early endosomes, lysosomes, or antigen-processing vesicles. Live cell microscopy demonstrated that the HIV-containing compartment was rapidly polarized toward the infectious synapse after contact with a T cell; however, the bulk of the concentrated virus remained in the DCs after T cell engagement. Individual virions were observed emerging from the compartment and fusing with the T cell membrane at the infectious synapse. The compartmentalized HIV, although engulfed by the cytoplasm, was fully accessible to HIV envelope-specific inhibitors and other membrane-impermeable probes that were delivered to the cell surface. These results demonstrate that HIV resides in an invaginated domain within DCs that is both contiguous with the plasma membrane and distinct from endocytic vesicles. We conclude that HIV virions are routed through this specialized compartment, which allows individual particles to be delivered to T cells during trans-infection.
Author Summary
Dendritic cells (DCs) patrol mucosal areas of the body, where they engulf invading pathogens and transport them to immune tissues. There the DCs degrade the microbes and present antigenic peptides to T lymphocytes to elicit specific immune responses. HIV-1 has appropriated this feature of the immune system to better establish and maintain infection of its primary target–CD4-positive T cells. DCs efficiently bind and degrade HIV, however a portion of the virus remains intact and can be transmitted into CD4 T cells, a process called trans-infection. DC maturation by various stimuli dramatically increases their capacity to trans-infect. Here we report that mature DCs concentrate infectious HIV into a pocket-like compartment that resides within the cell but remains physically connected to the cell surface. This structure is distinct from the intracellular degradative compartments that are used for microbial processing and presentation. The intact viral particles are retained within this compartment for extended periods, and individual particles can emerge and infect T cells at the cellular interface. We hypothesize that DCs form this compartment to sequester HIV off of the cell surface, however escape of virions from the pocket results in efficient infection of T cells during immune presentation events.
doi:10.1371/journal.ppat.1000134
PMCID: PMC2515344  PMID: 18725936
16.  A Role for microRNA-155 Modulation in the Anti-HIV-1 Effects of Toll-Like Receptor 3 Stimulation in Macrophages 
PLoS Pathogens  2012;8(9):e1002937.
HIV-1 infection of macrophages plays a key role in viral pathogenesis and progression to AIDS. Polyinosine-polycytidylic acid (poly(I∶C); a synthetic analog of dsRNA) and bacterial lipopolysaccharide (LPS), the ligands for Toll-like receptors (TLR) TLR3 and TLR4, respectively, are known to decrease HIV-1 infection in monocyte-derived macrophages (MDMs), but the mechanism(s) are incompletely understood. We found that poly(I∶C)- and LPS-stimulation of MDMs abrogated infection by CCR5-using, macrophage-tropic HIV-1, and by vesicular stomatitis virus glycoprotein-pseudotyped HIV-1 virions, while TLR2, TLR7 or TLR9 agonists only partially reduced infection to varying extent. Suppression of infection, or lack thereof, did not correlate with differential effects on CD4 or CCR5 expression, type I interferon induction, or production of pro-inflammatory cytokines or β-chemokines. Integrated pro-viruses were readily detected in unstimulated, TLR7- and TLR9-stimulated cells, but not in TLR3- or TLR4-stimulated MDMs, suggesting the alteration of post-entry, pre-integration event(s). Using microarray analysis and quantitative reverse transcription (RT)-PCR, we found increased microRNA (miR)-155 levels in MDMs upon TLR3/4- but not TLR7-stimulation, and a miR-155 specific inhibitor (but not a scrambled control) partially restored infectivity in poly(I∶C)-stimulated MDMs. Ectopic miR-155 expression remarkably diminished HIV-1 infection in primary MDMs and cell lines. Furthermore, poly(I∶C)-stimulation and ectopic miR-155 expression did not alter detection of early viral RT products, but both resulted in an accumulation of late RT products and in undetectable or extremely low levels of integrated pro-viruses and 2-LTR circles. Reduced mRNA and protein levels of several HIV-1 dependency factors involved in trafficking and/or nuclear import of pre-integration complexes (ADAM10, TNPO3, Nup153, LEDGF/p75) were found in poly(I∶C)-stimulated and miR-155-transfected MDMs, and a reporter assay suggested they are authentic miR-155 targets. Our findings provide evidence that miR-155 exerts an anti-HIV-1 effect by targeting several HIV-1 dependency factors involved in post-entry, pre-integration events, leading to severely diminished HIV-1 infection.
Author Summary
The infection of macrophages by HIV-1 is a crucial event in the pathogenesis of AIDS. Toll-like receptors (TLR) are a family of receptors present in macrophages – among other cells – that detect various components of microbes and trigger host defenses. It is known that stimulation of macrophages through TLR3 or TLR4 reduces their susceptibility to HIV-1 infection, but the mechanism is not well understood. Here we show for the first time in primary human macrophages that TLR3 and TLR4 but not other TLRs induce higher levels of microRNA-155 – a key regulator of inflammatory and immune responses – and that microRNA-155 has a remarkable anti-HIV-1 effect. MicroRNAs are small, non-coding RNAs that bind to target mRNAs based on sequence complementarity, and lead to reduced protein output. We also show that the anti-HIV-1 effects of microRNA-155 seem to be mediated through targeting the mRNAs of several cellular proteins needed by the virus for trafficking and/or nuclear import of the viral DNA, which is required for integration into the host DNA and successful infection. These studies provide evidence of novel microRNA-155 targets and may serve as the basis for an innovative approach to reduce cellular susceptibility to HIV-1 infection.
doi:10.1371/journal.ppat.1002937
PMCID: PMC3447756  PMID: 23028330
17.  Macrophages in Vaginal but Not Intestinal Mucosa Are Monocyte-Like and Permissive to Human Immunodeficiency Virus Type 1 Infection▿  
Journal of Virology  2009;83(7):3258-3267.
Mucosal surfaces play a major role in human immunodeficiency virus type 1 (HIV-1) transmission and pathogenesis, and yet the role of lamina propria macrophages in mucosal HIV-1 infection has received little investigative attention. We report here that vaginal and intestinal macrophages display distinct phenotype and HIV-1 permissiveness profiles. Vaginal macrophages expressed the innate response receptors CD14, CD89, CD16, CD32, and CD64 and the HIV-1 receptor/coreceptors CD4, CCR5, and CXCR4, similar to monocytes. Consistent with this phenotype, green fluorescent protein-tagged R5 HIV-1 entered macrophages in explanted vaginal mucosa as early as 30 min after inoculation of virus onto the epithelium, and purified vaginal macrophages supported substantial levels of HIV-1 replication by a panel of highly macrophage-tropic R5 viruses. In sharp contrast, intestinal macrophages expressed no detectable, or very low levels of, innate response receptors and HIV-1 receptor/coreceptors and did not support HIV-1 replication, although virus occasionally entered macrophages in intestinal tissue explants. Thus, vaginal, but not intestinal, macrophages are monocyte-like and permissive to R5 HIV-1 after the virus has translocated across the epithelium. These findings suggest that genital and gut macrophages have different roles in mucosal HIV-1 pathogenesis and that vaginal macrophages play a previously underappreciated but potentially important role in mucosal HIV-1 infection in the female genital tract.
doi:10.1128/JVI.01796-08
PMCID: PMC2655566  PMID: 19153236
18.  Impact of Round-the-Clock, Rapid Oral Fluid HIV Testing of Women in Labor in Rural India 
PLoS Medicine  2008;5(5):e92.
Background
Testing pregnant women for HIV at the time of labor and delivery is the last opportunity for prevention of mother-to-child HIV transmission (PMTCT) measures, particularly in settings where women do not receive adequate antenatal care. However, HIV testing and counseling of pregnant women in labor is a challenge, especially in resource-constrained settings. In India, many rural women present for delivery without any prior antenatal care. Those who do get antenatal care are not always tested for HIV, because of deficiencies in the provision of HIV testing and counseling services. In this context, we investigated the impact of introducing round-the-clock, rapid, point-of-care HIV testing and counseling in a busy labor ward at a tertiary care hospital in rural India.
Methods and Findings
After they provided written informed consent, women admitted to the labor ward of a rural teaching hospital in India were offered two rapid tests on oral fluid and finger-stick specimens (OraQuick Rapid HIV-1/HIV-2 tests, OraSure Technologies). Simultaneously, venous blood was drawn for conventional HIV ELISA testing. Western blot tests were performed for confirmatory testing if women were positive by both rapid tests and dual ELISA, or where test results were discordant. Round-the-clock (24 h, 7 d/wk) abbreviated prepartum and extended postpartum counseling sessions were offered as part of the testing strategy. HIV-positive women were administered PMTCT interventions. Of 1,252 eligible women (age range 18 y to 38 y) approached for consent over a 9 mo period in 2006, 1,222 (98%) accepted HIV testing in the labor ward. Of these, 1,003 (82%) women presented with either no reports or incomplete reports of prior HIV testing results at the time of admission to the labor ward. Of 1,222 women, 15 were diagnosed as HIV-positive (on the basis of two rapid tests, dual ELISA and Western blot), yielding a seroprevalence of 1.23% (95% confidence interval [CI] 0.61%–1.8%). Of the 15 HIV test–positive women, four (27%) had presented with reported HIV status, and 11 (73%) new cases of HIV infection were detected due to rapid testing in the labor room. Thus, 11 HIV-positive women received PMTCT interventions on account of round-the-clock rapid HIV testing and counseling in the labor room. While both OraQuick tests (oral and finger-stick) were 100% specific, one false-negative result was documented (with both oral fluid and finger-stick specimens). Of the 15 HIV-infected women who delivered, 13 infants were HIV seronegative at birth and at 1 and 4 mo after delivery; two HIV-positive infants died within a month of delivery.
Conclusions
In a busy rural labor ward setting in India, we demonstrated that it is feasible to introduce a program of round-the-clock rapid HIV testing, including prepartum and extended postpartum counseling sessions. Our data suggest that the availability of round-the-clock rapid HIV testing resulted in successful documentation of HIV serostatus in a large proportion (82%) of rural women who were unaware of their HIV status when admitted to the labor room. In addition, 11 (73%) of a total of 15 HIV-positive women received PMTCT interventions because of round-the-clock rapid testing in the labor ward. These findings are relevant for PMTCT programs in developing countries.
Nitika Pant Pai and colleagues report the results of offering a round-the-clock rapid HIV testing program in a rural labor ward setting in India.
Editors' Summary
Background.
Since the first reported case of AIDS (acquired immunodeficiency syndrome) in 1981, the number of people infected with the human immunodeficiency virus (HIV), which causes AIDS, has risen steadily. Now, more than 33 million people are infected, almost half of them women. HIV is most often spread through unprotected sex with an infected partner, but mother-to-child transmission (MTCT) of HIV is also an important transmission route. HIV-positive women often pass the virus to their babies during pregnancy, labor and delivery, and breastfeeding, if nothing is done to prevent viral transmission. In developed countries, interventions such as voluntary testing and counseling, safe delivery practices (for example, offering cesarean delivery to HIV-positive women), and antiretroviral treatment of the mother during pregnancy and labor and of her newborn baby have minimized the risk of MTCT. In developing countries, the prevention of MTCT (PMTCT) is much less effective, in part because pregnant women often do not know their HIV status. Consequently, in 2007, nearly half a million children became infected with HIV mainly through MTCT.
Why Was This Study Done?
In many developing countries, women do not receive adequate antenatal care. In India, for example, nearly half the women living in rural areas do not receive any antenatal care until they are in labor. This gives health care providers very little time in which to counsel women about HIV infection, test them for the virus, and start interventions to prevent MTCT. Furthermore, testing pregnant women in labor for HIV and counseling them is a challenge, particularly where resources are limited. In this study, therefore, the researchers investigate the feasibility and impact of introducing round-the-clock, rapid HIV testing and counseling in a busy labor ward in a rural teaching hospital in Sevagram, India.
What Did the Researchers Do and Find?
Women admitted to the labor ward between January and September 2006 were offered two rapid HIV tests—one that used a saliva sample and the other that used blood taken from a finger prick. Blood was also taken from a vein for conventional HIV testing. All the women were given a 15-minute counseling session about how HIV is transmitted, the importance of HIV testing, and information on PMTCT before their child was born (prepartum counseling), and a longer postpartum counseling session. HIV-positive women were given a cesarean delivery where possible and antiretroviral drug treatment to reduce MTCT. 1,222 women admitted to the labor ward during the study period (1,003 of whom did not know their HIV status) accepted HIV testing. Of 15 study participants who were HIV positive, 11 learnt of their HIV status in the labor room. Two babies born to these HIV-positive women were HIV positive and died within a month of delivery; the other 13 babies were HIV negative at birth and at 1 and 4 months after delivery. Finally, the rapid HIV tests missed only one HIV-positive woman (no false-positive results were given), and the time from enrolling a woman into the study through referring her for PMTCT intervention where necessary averaged 40–60 minutes.
What Do These Findings Mean?
These findings show the feasibility and positive impact of the introduction of round-the-clock pre- and postpartum HIV counseling and rapid HIV testing into a busy rural Indian labor ward. Few of the women entering this ward knew their HIV status previously but the introduction of these facilities in this setting successfully informed these women of their HIV status. In addition, the round-the-clock counseling and testing led to 11 women and their babies receiving PMTCT interventions who would otherwise have been missed. These findings need to be confirmed in other settings and the cost-effectiveness and sustainability of this approach for the improvement of PMTCT in developing countries needs to be investigated. Nevertheless, these findings suggest that round-the-clock rapid HIV testing might be an effective and acceptable way to reduce MTCT of HIV in many developing countries.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050092.
Read a related PLoS Medicine Perspective article
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS and on HIV infection in women
HIV InSite has comprehensive information on all aspects of HIV/AIDS
Women, Children, and HIV provides extensive information on the prevention of mother-to-child transmission of HIV in developing countries
Information is available from Avert, an international AIDS charity, on HIV and AIDS in India, on women, HIV, and AIDS, and on HIV and AIDS prevention, including the prevention of mother-to-child transmission
AIDSinfo, a service of the US Department of Health and Human Services provides health information for HIV-positive pregnant women (in English and Spanish)
doi:10.1371/journal.pmed.0050092
PMCID: PMC2365974  PMID: 18462011
19.  Induction of Keratinocyte Growth Factor 1 Expression by Lipopolysaccharide Is Regulated by CD-14 and Toll-Like Receptors 2 and 4  
Infection and Immunity  2002;70(12):6541-6548.
Periodontal disease is a chronic inflammatory condition that is associated with increased concentrations of gram-negative pathogenic bacteria and epithelial cell proliferation. Regulation of this proliferation is poorly understood but is most likely controlled by locally expressed growth factors. Keratinocyte growth factor 1, an epithelium-specific growth factor, is expressed by gingival fibroblasts, and its expression is regulated in a concentration-dependent manner by lipopolysaccharide. In this study, induction of keratinocyte growth factor 1 protein expression was dependent on gingival fibroblast expression of membrane CD14 (mCD14) and Toll-like receptors 2 and 4. Lipopolysaccharides from Escherichia coli and Porphyromonas gingivalis induced membrane expression of CD14 at 1, 3, and 24 h. Specifically, lipopolysaccharide induced low mCD14 expression gingival fibroblasts to express mCD14 at a level consistent with that of high mCD14 expression cells. Functional studies with specific blocking antibodies for CD14 and Toll-like receptors 2 and 4 implicated all of these molecules in signal transduction. The rapid decrease in cell membrane expression of Toll-like receptors 2 and 4 after treatment with lipopolysaccharide was consistent with receptor internalization, and blocking of either of these receptors completely inhibited keratinocyte growth factor 1 protein expression. The transcription factors AP-1 and NF-κB were involved in lipopolysaccharide induction of keratinocyte growth factor 1 mRNA and protein expression. These results suggest that lipopolysaccharide may induce proliferation of periodontal epithelial cells by upregulating keratinocyte growth factor 1 expression via the CD14 and Toll-like receptor signaling pathway.
doi:10.1128/IAI.70.12.6541-6548.2002
PMCID: PMC132971  PMID: 12438323
20.  HIV-1 Transmission during Early Infection in Men Who Have Sex with Men: A Phylodynamic Analysis 
PLoS Medicine  2013;10(12):e1001568.
Erik Volz and colleagues use HIV genetic information from a cohort of men who have sex with men in Detroit, USA to dissect the timing of onward transmission during HIV infection.
Please see later in the article for the Editors' Summary
Background
Conventional epidemiological surveillance of infectious diseases is focused on characterization of incident infections and estimation of the number of prevalent infections. Advances in methods for the analysis of the population-level genetic variation of viruses can potentially provide information about donors, not just recipients, of infection. Genetic sequences from many viruses are increasingly abundant, especially HIV, which is routinely sequenced for surveillance of drug resistance mutations. We conducted a phylodynamic analysis of HIV genetic sequence data and surveillance data from a US population of men who have sex with men (MSM) and estimated incidence and transmission rates by stage of infection.
Methods and Findings
We analyzed 662 HIV-1 subtype B sequences collected between October 14, 2004, and February 24, 2012, from MSM in the Detroit metropolitan area, Michigan. These sequences were cross-referenced with a database of 30,200 patients diagnosed with HIV infection in the state of Michigan, which includes clinical information that is informative about the recency of infection at the time of diagnosis. These data were analyzed using recently developed population genetic methods that have enabled the estimation of transmission rates from the population-level genetic diversity of the virus. We found that genetic data are highly informative about HIV donors in ways that standard surveillance data are not. Genetic data are especially informative about the stage of infection of donors at the point of transmission. We estimate that 44.7% (95% CI, 42.2%–46.4%) of transmissions occur during the first year of infection.
Conclusions
In this study, almost half of transmissions occurred within the first year of HIV infection in MSM. Our conclusions may be sensitive to un-modeled intra-host evolutionary dynamics, un-modeled sexual risk behavior, and uncertainty in the stage of infected hosts at the time of sampling. The intensity of transmission during early infection may have significance for public health interventions based on early treatment of newly diagnosed individuals.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Since the first recorded case of AIDS in 1981, the number of people infected with HIV, the virus that causes AIDS, has risen steadily. About 34 million people are currently HIV-positive, and about 2.5 million people become newly infected with HIV every year. Because HIV is usually transmitted through unprotected sex with an infected partner, individuals can reduce their risk of infection by abstaining from sex, by having only one or a few partners, and by always using condoms. Most people do not become ill immediately after infection with HIV, although some develop a short flu-like illness. The next stage of HIV infection, which may last more than ten years, also has no major symptoms, but during this stage, HIV slowly destroys immune system cells. Eventually, the immune system can no longer fight off infections by other disease-causing organisms, and HIV-positive people then develop one or more life-threatening AIDS-defining conditions, including unusual infections and specific types of cancer. HIV infection can be controlled, but not cured, by taking a daily cocktail of antiretroviral drugs.
Why Was This Study Done?
The design of effective programs to prevent the spread of HIV/AIDS depends on knowing how HIV transmissibility varies over the course of HIV infection. Consider, for example, a prevention strategy that focuses on increasing treatment rates: antiretroviral drugs, in addition to reducing illness and death among HIV-positive people, reduce HIV transmission from HIV-positive individuals. “Treatment as prevention” can only block transmissions that occur after diagnosis and entry into care. However, the transmissibility of HIV per sexual contact depends on a person's viral load, which peaks during early HIV infection, when people are often unaware of their HIV status and may still be following the high-risk patterns of sexual behavior that caused their own infection. Epidemiological surveillance data (information on HIV infections within populations) can be used to estimate how many new HIV infections occur within a population annually (HIV incidence) and the proportion of the population that is HIV-positive (HIV prevalence), but cannot be used to estimate the timing of transmission events. In this study, the researchers use “phylodynamic analysis” to estimate HIV incidence and prevalence and the timing of HIV transmission during infection. HIV, like many other viruses, rapidly accumulates genetic changes. The timing of transmission influences the pattern of these changes. Viral phylodynamic analysis—the quantitative study of how epidemiological, immunological, and evolutionary processes shape viral phylogenies (evolutionary trees)—can therefore provide estimates of transmission dynamics.
What Did the Researchers Do and Find?
The researchers obtained HIV sequence data (collected for routine surveillance of antiretroviral resistance mutations) and epidemiological surveillance data (including information on the stage of infection at diagnosis) for 662 HIV-positive men who have sex with men living in the Detroit metropolitan area of Michigan. They constructed a phylogenetic tree from the sequences using a “relaxed clock” approach and then fitted an epidemiological model (a mathematical model that represents the progress of individual patients through various stages of HIV infection) to the sequence data. Their approach, which integrates surveillance data and genetic data, yielded estimates of HIV incidence and prevalence among the study population similar to those obtained from surveillance data alone. However, it also provided information about HIV transmission that could not be obtained from surveillance data alone. In particular, it allowed the researchers to estimate that, in the current HIV epidemic among men who have sex with men in Detroit, 44.7% of HIV transmissions occur during the first year of infection.
What Do These Findings Mean?
The robustness of these findings depends on the validity of the assumptions included in the researchers' population genetic model and on the accuracy of the data fed into the model, and may not be generalizable to other cities or to other risk groups. Nevertheless, the findings of this analysis, which can be repeated in any setting where HIV sequence data for individual patients can be linked to patient-specific clinical and behavioral information, have important implications for HIV control strategies based on the early treatment of newly diagnosed individuals. Because relatively few infected individuals are diagnosed during early HIV infection, when the HIV transmission rate is high, it is unlikely, suggest the researchers, that the “treatment as prevention” strategy will effectively control the spread of HIV unless there are very high rates of HIV testing and treatment.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001568.
This study is further discussed in a PLOS Medicine Perspective by Timothy Hallett
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
NAM/aidsmap provides basic information about HIV/AIDS and summaries of recent research findings on HIV care and treatment
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on HIV treatment as prevention (in English and Spanish)
The PLOS Medicine Collection Investigating the Impact of Treatment on New HIV Infections provides more information about HIV treatment as prevention
A PLOS Computational Biology Topic Page (a review article that is a published copy of record of a dynamic version of the article as found in Wikipedia) about viral phylodynamics is available
The US National Institute of Health–funded HIV Sequence Database contains HIV sequences and tools to analyze these sequences
Patient stories about living with HIV/AIDS are available through Avert; the charity website Healthtalkonline also provides personal stories about living with HIV
doi:10.1371/journal.pmed.1001568
PMCID: PMC3858227  PMID: 24339751
21.  Differential Tropism and Chemokine Receptor Expression of Human Immunodeficiency Virus Type 1 in Neonatal Monocytes, Monocyte-Derived Macrophages, and Placental Macrophages 
Journal of Virology  1998;72(2):1334-1344.
Laboratory-adapted (LA) macrophage-tropic (M-tropic) human immunodeficiency virus type 1 (HIV-1) isolates (e.g., HIV-1Ba-L) and low-passage primary (PR) isolates differed markedly in tropism for syngeneic neonatal monocytes, monocyte-derived macrophages (MDMs), and placental macrophages (PMs). Newly adherent neonatal monocytes and cultured PMs were highly refractory to infection with PR HIV-1 isolates yet were permissive for LA M-tropic isolates. Day 4 MDMs were also permissive for LA M-tropic isolates and additionally, were permissive for over half the PR isolates tested. Qualitative differences in PR HIV-1 infection of monocytes/MDMs could not be correlated with CD4 levels alone, and in all three cell types the block to PR HIV-1 strain replication preceded reverse transcription. Neonatal monocyte susceptibility to PR HIV-1 strains correlated with increasing CCR-5 expression during maturation. CCR-5 could not be detected on newly adherent (day 1) neonatal monocytes, in contrast to adult monocytes (H. Naif et al., J. Virol. 72:830–836, 1998), but was readily detectable after 4 to 7 days of culture. However, moderate CCR-5 mRNA levels were present in day 1 neonatal monocytes and remained constant during monocyte maturation. CCR-5 was not detectable on the surface of PMs, yet the receptor was present within permeabilized cells. Notably, two brain-derived PR HIV-1 isolates from a single patient, differing in their V3 loops, were discordant in their abilities to infect neonatal monocytes/MDMs and PMs, yet both isolates could infect newly adherent adult monocytes. Together these data strongly suggest that LA HIV-1 isolates are able to infect neonatal monocytes at earlier stages of maturation and lower-level expression of CCR-5 than PR isolates. The differences between neonatal and adult monocytes in susceptibility to PR isolates may also be related to the level of CCR-5 expression.
PMCID: PMC124612  PMID: 9445034
22.  The Colocalization Potential of HIV-Specific CD8+ and CD4+ T-Cells is Mediated by Integrin β7 but Not CCR6 and Regulated by Retinoic Acid 
PLoS ONE  2012;7(3):e32964.
CD4+ T-cells from gut-associated lymphoid tissues (GALT) are major targets for HIV-1 infection. Recruitment of excess effector CD8+ T-cells in the proximity of target cells is critical for the control of viral replication. Here, we investigated the colocalization potential of HIV-specific CD8+ and CD4+ T-cells into the GALT and explored the role of retinoic acid (RA) in regulating this process in a cohort of HIV-infected subjects with slow disease progression. The expression of the gut-homing molecules integrin β7, CCR6, and CXCR3 was identified as a “signature” for HIV-specific but not CMV-specific CD4+ T-cells thus providing a new explanation for their enhanced permissiveness to infection in vivo. HIV-specific CD8+ T-cells also expressed high levels of integrin β7 and CXCR3; however CCR6 was detected at superior levels on HIV-specific CD4+ versus CD8+ T-cells. All trans RA (ATRA) upregulated the expression of integrin β7 but not CCR6 on HIV-specific T-cells. Together, these results suggest that HIV-specific CD8+ T-cells may colocalize in excess with CD4+ T-cells into the GALT via integrin β7 and CXCR3, but not via CCR6. Considering our previous findings that CCR6+CD4+ T-cells are major cellular targets for HIV-DNA integration in vivo, a limited ability of CD8+ T-cells to migrate in the vicinity of CCR6+CD4+ T-cells may facilitate HIV replication and dissemination at mucosal sites.
doi:10.1371/journal.pone.0032964
PMCID: PMC3314661  PMID: 22470433
23.  Human Immunodeficiency Virus Type 1 Stimulates the Expression and Production of Secretory Leukocyte Protease Inhibitor (SLPI) in Oral Epithelial Cells: a Role for SLPI in Innate Mucosal Immunity 
Journal of Virology  2005;79(10):6432-6440.
The innate immune response is a key barrier against pathogenic microorganisms such as human immunodeficiency virus type 1 (HIV-1). Because HIV-1 is rarely transmitted orally, we hypothesized that oral epithelial cells participate in the innate immune defense against this virus. We further hypothesized that secretory leukocyte protease inhibitor (SLPI), a 12-kDa mucosal antiviral protein, is a component of the host immune response to this virus. Here we demonstrated constitutive expression and production of SLPI in immortalized human oral keratinocytes. Brief exposure of cells to HIV-1 BaL and HXB2 significantly increased SLPI mRNA and protein production compared to that in mock-exposed cells (P < 0.01), as evaluated by real-time quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay. HIV-1-mediated stimulation of SLPI occurred at the transcriptional level, was dose and time dependent, was elicited by heat-inactivated and infectious viruses, and did not depend on cellular infection. Experiments with purified retroviral proteins showed that the stimulatory effect was induced specifically by external envelope glycoproteins from HIV-1 and simian immunodeficiency virus. SLPI responsiveness to HIV-1 was also observed in an unrelated oral epithelial cell line and in normal (nonimmortalized) human oral epithelial cells isolated from healthy uninfected gingival tissues. In this first report of SLPI regulation by HIV-1, we show that the expression and production of the antimicrobial and anti-inflammatory protein can be stimulated in oral epithelial cells by the virus through interactions with gp120 in the absence of direct infection. These findings indicate that SLPI is a component of the oral mucosal response to HIV-1.
doi:10.1128/JVI.79.10.6432-6440.2005
PMCID: PMC1091668  PMID: 15858026
24.  HIV-1 Reactivation Induced by the Periodontal Pathogens Fusobacterium nucleatum and Porphyromonas gingivalis Involves Toll-Like Receptor 4 and 9 Activation in Monocytes/Macrophages▿  
Although oral coinfections (e.g., periodontal disease) are highly prevalent in human immunodeficiency virus type 1-positive (HIV-1+) patients and appear to positively correlate with viral load levels, the potential for oral bacteria to induce HIV-1 reactivation in latently infected cells has received little attention. We showed that HIV-1 long terminal repeat (LTR) promoter activation can be induced by periodontopathogens in monocytes/macrophages; nevertheless, the mechanisms involved in this response remain undetermined. Since Toll-like receptor 2 (TLR2), TLR4, and TLR9 activation have been involved in HIV-1 recrudescence, we sought to determine the role of these TLRs in HIV-1 reactivation induced by the periodontal pathogens Fusobacterium nucleatum and Porphyromonas gingivalis using BF24 monocytes/macrophages stably transfected with the HIV-1 promoter driving chloramphenicol acetyltransferase (CAT) expression and THP89GFP cells, a model of HIV-1 latency. We demonstrated that TLR9 activation by F. nucleatum and TLR2 activation by both bacteria appear to be involved in HIV-1 reactivation; however, TLR4 activation had no effect. Moreover, the autocrine activity of tumor necrosis factor alpha (TNF-α) but not interleukin-1β (IL-1β) produced in response to bacteria could impact viral reactivation. The transcription factors NF-κB and Sp1 appear to be positively regulating HIV-1 reactivation induced by these oral pathogens. These results suggest that oral Gram-negative bacteria (F. nucleatum and P. gingivalis) associated with oral and systemic chronic inflammatory disorders enhance HIV-1 reactivation in monocytes/macrophages through TLR2 and TLR9 activation in a mechanism that appears to be transcriptionally regulated. Increased bacterial growth and emergence of these bacteria or their products accompanying chronic oral inflammatory diseases could be risk modifiers for viral replication, systemic immune activation, and AIDS progression in HIV-1+ patients.
doi:10.1128/CVI.00009-10
PMCID: PMC2944464  PMID: 20610663
25.  Antigen Load and Viral Sequence Diversification Determine the Functional Profile of HIV-1–Specific CD8+ T Cells 
PLoS Medicine  2008;5(5):e100.
Background
Virus-specific CD8+ T lymphocytes play a key role in the initial reduction of peak viremia during acute viral infections, but display signs of increasing dysfunction and exhaustion under conditions of chronic antigen persistence. It has been suggested that virus-specific CD8+ T cells with a “polyfunctional” profile, defined by the capacity to secrete multiple cytokines or chemokines, are most competent in controlling viral replication in chronic HIV-1 infection. We used HIV-1 infection as a model of chronic persistent viral infection to investigate the process of exhaustion and dysfunction of virus-specific CD8+ T cell responses on the single-epitope level over time, starting in primary HIV-1 infection.
Methods and Findings
We longitudinally analyzed the polyfunctional epitope-specific CD8+ T cell responses of 18 patients during primary HIV-1 infection before and after therapy initiation or sequence variation in the targeted epitope. Epitope-specific CD8+ T cells responded with multiple effector functions to antigenic stimulation during primary HIV-1 infection, but lost their polyfunctional capacity in response to antigen and up-regulated programmed death 1 (PD-1) expression with persistent viremic infection. This exhausted phenotype significantly decreased upon removal of stimulation by antigen, either in response to antiretroviral therapy or by reduction of epitope-specific antigen load in the presence of ongoing viral replication, as a consequence of in vivo selection of cytotoxic T lymphocyte escape mutations in the respective epitopes. Monofunctionality increased in CD8+ T cell responses directed against conserved epitopes from 49% (95% confidence interval 27%–72%) to 76% (56%–95%) (standard deviation [SD] of the effect size 0.71), while monofunctionality remained stable or slightly decreased for responses directed against escaped epitopes from 61% (47%–75%) to 56% (42%–70%) (SD of the effect size 0.18) (p < 0.05).
Conclusion
These data suggest that persistence of antigen can be the cause, rather than the consequence, of the functional impairment of virus-specific T cell responses observed during chronic HIV-1 infection, and underscore the importance of evaluating autologous viral sequences in studies aimed at investigating the relationship between virus-specific immunity and associated pathogenesis.
Marcus Altfeld and colleagues suggest that the exhaustion of virus-specific CD8+ T cells during chronic HIV infection likely results from the persistence of antigen.
Editors' Summary
Background.
Viruses are small infectious agents responsible for many human diseases, including acquired immunodeficiency syndrome (AIDS). Like other viruses, the human immunodeficiency virus 1 (HIV-1; the cause of AIDS) enters human cells and uses the cellular machinery to replicate before bursting out of its temporary home. During the initial stage of HIV infection, a particular group of cells in the human immune system, CD8+ T cells, are thought to be important in controlling the level of the virus. These immune system cells recognize pieces of viral protein called antigens displayed on the surface of infected cells; different subsets of CD8+ T cells recognize different antigens. When a CD8+ T cell recognizes its specific antigen (or more accurately, a small part of the antigen called an “epitope”), it releases cytotoxins (which kill the infected cells) and cytokines, proteins that stimulate CD8+ T cell proliferation and activate other parts of the immune system. With many viruses, when a person first becomes infected (an acute viral infection), antigen-specific CD8+ T cells completely clear the infection. But with HIV-1 and some other viruses, these cells do not manage to remove all the viruses from the body and a chronic (long-term) infection develops, during which the immune system is constantly exposed to viral antigen.
Why Was This Study Done?
In HIV-1 infections (and other chronic viral infections), virus-specific CD8+ T cells lose their ability to proliferate, to make cytokines, and to kill infected cells as patients progress to the long-term stages of infection. That is, the virus-specific CD8+ T cells gradually lose their “effector” functions and become functionally impaired or “exhausted.” “Polyfunctional” CD8+ T cells (those that release multiple cytokines in response to antigen) are believed to be essential for an effective CD8+ T cell response, so scientists trying to develop HIV-1 vaccines would like to stimulate the production of this type of cell. To do this they need to understand why these polyfunctional cells are lost during chronic infections. Is their loss the cause or the result of viral persistence? In other words, does the constant presence of viral antigen lead to the exhaustion of CD8+ T cells during chronic HIV infection? In this study, the researchers investigate this question by looking at the polyfunctionality of CD8+ cells responding to several different viral epitopes at various times during HIV-1 infection, starting very early after infection with HIV-1 had occurred.
What Did the Researchers Do and Find?
The researchers enrolled 18 patients recently infected with HIV-1 and analyzed their CD8+ T cell responses to specific epitopes at various times after enrollment using a technique called flow cytometry. They found that the epitope-specific CD8+ cells produced several effector proteins after antigen stimulation during the initial stage of HIV-1 infection, but lost their polyfunctionality in the face of persistent viral infection. The CD8+ T cells also increased their production of programmed death 1 (PD-1), a protein that has been shown to be associated with the functional impairment of CD8+ T cells. Some of the patients began antiretroviral therapy during the study, and the researchers found that this treatment, which reduced the viral load, reversed CD8+ T cell exhaustion. Finally, the appearance in the patients' blood of viruses that had made changes in the specific epitopes recognized by the CD8+ T cells to avoid being killed by these cells, also reversed the exhaustion of the T cells recognizing these particular epitopes.
What Do These Findings Mean?
These findings suggest that the constant presence of HIV-1 antigen causes the functional impairment of virus-specific CD8+ T cell responses during chronic HIV-1 infections. Treatment with antiretroviral drugs reversed this functional impairment by reducing the amount of antigen in the patients. Similarly, the appearance of viruses with altered epitopes, which effectively reduced the amount of antigen recognized by those epitope-specific CD8+ T cells without reducing the viral load, also reversed T cell exhaustion. These results would not have been seen if the functional impairment of CD8+ cells were the cause rather than the result of antigen persistence. By providing new insights into how the T cell response to viruses evolves during persistent viral infections, these findings should help in the design of vaccines against HIV and other viruses that cause chronic viral infections.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050100.
Read a related PLoS Medicine Research in Translation article
Learn more from the researchers' Web site, the Partners AIDS Research Center
Wikipedia has a page on cytotoxic T cells (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIV InSite has comprehensive information on all aspects of HIV/AIDS, including a detailed article on the immunopathogenesis of HIV infection
NAM, a UK registered charity, provides information about all aspects of HIV and AIDS, including a fact sheet on the stages of HIV infection and on the immune response to HIV
Information is available from Avert, an international AIDS charity, on all aspects of HIV/AIDS, including information on the stages of HIV infection
doi:10.1371/journal.pmed.0050100
PMCID: PMC2365971  PMID: 18462013

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