A hunter virus that targets both infected cells and HIV free virions: Implications for therapy

The design of ‘hunter’ viruses aimed at destroying human immunodeficiency virus (HIV) infected cells is an active area of research that has produced promising results in vitro. Hunters are designed to target exposed viral envelope proteins in the membranes of infected cells, but there is evidence that the hunter may also target envelope proteins of free HIV, inducing virus-virus fusion. In order to predict the effects of this fusion on therapy outcomes and determine whether fusion ability is advantageous for hunter virus design, we have constructed a model to account for the possibility of hunter-HIV fusion. The study was based on a target cell-limited model of HIV infection and it examined the hunter therapeutic effect on recovering the HIV main target cells, the activated CD4⁺ T lymphocytes. These cells assist in setting up an immune response to opportunistic infections. The study analyzed the hunter dual mechanisms to control infection and because of diverse estimates for viral production and clearance of HIV, simulations were examined at rates spanning an order of magnitude. Results indicate that without hunter-HIV fusion ability, hunters that kill HIV-infected cells lead to a substantial recovery of healthy cell population at both low and high HIV turnover rates. When hunter-HIV fusion is included, cell recovery was particularly enhanced at lower HIV turnover rates. This study shows that the fusion ability, in addition to hunter infection ability, could be a favorable attribute for improving the efficacy of hunter-viral therapy. These results provide support for the potential use of engineered viruses to control HIV and other viral infections.     

Antimicrobial peptides from amphibian skin potently inhibit human immunodeficiency virus infection and transfer of virus from dendritic cells to T cells

Topical antimicrobicides hold great promise in reducing human immunodeficiency virus (HIV) transmission. Amphibian skin provides a rich source of broad-spectrum antimicrobial peptides including some that have antiviral activity. We tested 14 peptides derived from diverse amphibian species for the capacity to inhibit HIV infection. Three peptides (caerin 1.1, caerin 1.9, and maculatin 1.1) completely inhibited HIV infection of T cells within minutes of exposure to virus at concentrations that were not toxic to target cells. These peptides also suppressed infection by murine leukemia virus but not by reovirus, a structurally unrelated nonenveloped virus. Preincubation with peptides prevented viral fusion to target cells and disrupted the HIV envelope. Remarkably, these amphibian peptides also were highly effective in inhibiting the transfer of HIV by dendritic cells (DCs) to T cells, even when DCs were transiently exposed to peptides 8 h after virus capture. These data suggest that amphibian-derived peptides can access DC-sequestered HIV and destroy the virus before it can be transferred to T cells. Thus, amphibian-derived antimicrobial peptides show promise as topical inhibitors of mucosal HIV transmission and provide novel tools to understand the complex biology of HIV capture by DCs.     

Fighting a virus with a virus: a dynamic model for HIV-1 therapy

A mathematical model examined a potential therapy for controlling viral infections using genetically modified viruses. The control of the infection is an indirect effect of the selective elimination by an engineered virus of infected cells that are the source of the pathogens. Therefore, this engineered virus could greatly compensate for a dysfunctional immune system compromised by AIDS. In vitro studies using engineered viruses have been shown to decrease the HIV-1 load about 1000-fold. However, the efficacy of this potential treatment for reducing the viral load in AIDS patients is unknown. The present model studied the interactions among the HIV-1 virus, its main host cell (activated CD4+ T cells), and a therapeutic engineered virus in an in vivo context; and it examined the conditions for controlling the pathogen. This model predicted a significant drop in the HIV-1 load, but the treatment does not eradicate HIV. A basic estimation using a currently engineered virus indicated an HIV-1 load reduction of 92% and a recovery of host cells to 17% of their normal level. Greater success (98% HIV reduction, 44% host cells recovery) is expected as more competent engineered viruses are designed. These results suggest that therapy using viruses could be an alternative to extend the survival of AIDS patients.     

Impaired Antibody Response Causes Persistence of Prototypic T Cell–Contained Virus

CD8 T cells are recognized key players in control of persistent virus infections, but increasing evidence suggests that assistance from other immune mediators is also needed. Here, we investigated whether specific antibody responses contribute to control of lymphocytic choriomeningitis virus (LCMV), a prototypic mouse model of systemic persistent infection. Mice expressing transgenic B cell receptors of LCMV-unrelated specificity, and mice unable to produce soluble immunoglobulin M (IgM) exhibited protracted viremia or failed to resolve LCMV. Virus control depended on immunoglobulin class switch, but neither on complement cascades nor on Fc receptor γ chain or Fc γ receptor IIB. Cessation of viremia concurred with the emergence of viral envelope-specific antibodies, rather than with neutralizing serum activity, and even early nonneutralizing IgM impeded viral persistence. This important role for virus-specific antibodies may be similarly underappreciated in other primarily T cell–controlled infections such as HIV and hepatitis C virus, and we suggest this contribution of antibodies be given consideration in future strategies for vaccination and immunotherapy.     

Turing Patterns from Dynamics of Early HIV Infection

We have developed a mathematical model for in-host virus dynamics that includes spatial chemotaxis and diffusion across a two-dimensional surface representing the vaginal or rectal epithelium at primary HIV infection. A linear stability analysis of the steady state solutions identified conditions for Turing instability pattern formation. We have solved the model equations numerically using parameter values obtained from previous experimental results for HIV infections. Simulations of the model for this surface show hot spots of infection. Understanding this localization is an important step in the ability to correctly model early HIV infection. These spatial variations also have implications for the development and effectiveness of microbicides against HIV.     

Capture and transfer of simian immunodeficiency virus by macaque dendritic cells is enhanced by DC-SIGN

Dendritic cells (DCs) are among the first cells encountered by human and simian immunodeficiency virus (HIV and SIV) following mucosal infection. Because these cells efficiently capture and transmit virus to T cells, they may play a major role in mediating HIV and SIV infection. Recently, a C-type lectin protein present on DCs, DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), was shown to efficiently bind and present HIV and SIV to CD4(+), coreceptor-positive cells in trans. However, the significance of DC-SIGN for virus transmission and pathogenesis in vivo remains unclear. Because SIV infection of macaques may represent the best model to study the importance of DC-SIGN in HIV infection, we cloned and characterized pig-tailed macaque DC-SIGN and generated monoclonal antibodies (MAbs) against it. We demonstrate that, like human DC-SIGN, pig-tailed macaque DC-SIGN (ptDC-SIGN) is expressed on DCs and macrophages but not on monocytes, T cells, or B cells. Moderate levels of ptDC-SIGN expression were detected on the surface of DCs, and low-level expression was found on macrophages. Additionally, we show that ptDC-SIGN efficiently binds and transmits replication-competent SIVmne variants to CD4(+), coreceptor-positive cells. Moreover, transmission of virus between pig-tailed macaque DCs and CD4(+) T cells is largely ptDC-SIGN dependent. Interestingly, MAbs directed against ptDC-SIGN vary in the capacity to block transmission of different SIVmne variants. These data demonstrate that ptDC-SIGN plays a central role in transmitting virus from macaque DCs to T cells, and they suggest that SIVmne variants may differ in their interactions with ptDC-SIGN. Thus, SIVmne infection of pig-tailed macaques may provide an opportunity to investigate the significance of DC-SIGN in primate lentiviral infections.     

Chlamydia trachomatis Infection of Endocervical Epithelial Cells Enhances Early HIV Transmission Events

Chlamydia trachomatis causes a predominantly asymptomatic, but generally inflammatory, genital infection that is associated with an increased risk for HIV acquisition. Endocervical epithelial cells provide the major niche for this obligate intracellular bacterium in women, and the endocervix is also a tissue in which HIV transmission can occur. The mechanism by which CT infection enhances HIV susceptibility at this site, however, is not well understood. Utilizing the A2EN immortalized endocervical epithelial cell line grown on cell culture inserts, we evaluated the direct role that CT-infected epithelial cells play in facilitating HIV transmission events. We determined that CT infection significantly enhanced the apical-to-basolateral migration of cell-associated, but not cell-free, HIV_BaL, a CCR5-tropic strain of virus, across the endocervical epithelial barrier. We also established that basolateral supernatants from CT-infected A2EN cells significantly enhanced HIV replication in peripheral mononuclear cells and a CCR5+ T cell line. These results suggest that CT infection of endocervical epithelial cells could facilitate both HIV crossing the mucosal barrier and subsequent infection or replication in underlying target cells. Our studies provide a mechanism by which this common STI could potentially promote the establishment of founder virus populations and the maintenance of local HIV reservoirs in the endocervix. Development of an HIV/STI co-infection model also provides a tool to further explore the role of other sexually transmitted infections in enhancing HIV acquisition.     

Multiploid inheritance of HIV-1 during cell-to-cell infection

During cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1), many viral particles can be simultaneously transferred from infected to uninfected CD4 T cells through structures called virological synapses (VS). Here we directly examine how cell-free and cell-to-cell infections differ from infections initiated with cell-free virus in the number of genetic copies that are transmitted from one generation to the next, i.e., the genetic inheritance. Following exposure to HIV-1-expressing cells, we show that target cells with high viral uptake are much more likely to become infected. Using T cells that coexpress distinct fluorescent HIV-1 variants, we show that multiple copies of HIV-1 can be cotransmitted across a single VS. In contrast to cell-free HIV-1 infection, which titrates with Poisson statistics, the titration of cell-associated HIV-1 to low rates of overall infection generates a constant fraction of the newly infected cells that are cofluorescent. Triple infection was also readily detected when cells expressing three fluorescent viruses were used as donor cells. A computational model and a statistical model are presented to estimate the degree to which cofluorescence underestimates coinfection frequency. Lastly, direct detection of HIV-1 proviruses using fluorescence in situ hybridization confirmed that significantly more HIV-1 DNA copies are found in primary T cells infected with cell-associated virus than in those infected with cell-free virus. Together, the data suggest that multiploid inheritance is common during cell-to-cell HIV-1 infection. From this study, we suggest that cell-to-cell infection may explain the high copy numbers of proviruses found in infected cells in vivo and may provide a mechanism through which HIV preserves sequence heterogeneity in viral quasispecies through genetic complementation.     

PD-L1 Expression on Retrovirus-Infected Cells Mediates Immune Escape from CD8+ T Cell Killing

Cytotoxic CD8+ T Lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. Signaling of the inhibitory receptor PD-1 is an important mechanism for the development of virus-specific CD8+ T cell dysfunction. However, it has recently been shown that during the initial phase of infection virus-specific CD8+ T cells express high levels of PD-1, but are fully competent in producing cytokines and killing virus-infected target cells. To better understand the role of the PD-1 signaling pathway in CD8+ T cell cytotoxicity during acute viral infections we analyzed the expression of the ligand on retrovirus-infected cells targeted by CTLs. We observed increased levels of PD-L1 expression after infection of cells with the murine Friend retrovirus (FV) or with HIV. In FV infected mice, virus-specific CTLs efficiently eliminated infected target cells that expressed low levels of PD-L1 or that were deficient for PD-L1 but the population of PD-L1high cells escaped elimination and formed a reservoir for chronic FV replication. Infected cells with high PD-L1 expression mediated a negative feedback on CD8+ T cells and inhibited their expansion and cytotoxic functions. These findings provide evidence for a novel immune escape mechanism during acute retroviral infection based on PD-L1 expression levels on virus infected target cells.     

Genetic Drift of HIV Populations in Culture

Populations of Human Immunodeficiency Virus type 1 (HIV-1) undergo a surprisingly large amount of genetic drift in infected patients despite very large population sizes, which are predicted to be mostly deterministic. Several models have been proposed to explain this phenomenon, but all of them implicitly assume that the process of virus replication itself does not contribute to genetic drift. We developed an assay to measure the amount of genetic drift for HIV populations replicating in cell culture. The assay relies on creation of HIV populations of known size and measurements of variation in frequency of a neutral allele. Using this assay, we show that HIV undergoes approximately ten times more genetic drift than would be expected from its population size, which we defined as the number of infected cells in the culture. We showed that a large portion of the increase in genetic drift is due to non-synchronous infection of target cells. When infections are synchronized, genetic drift for the virus is only 3-fold higher than expected from its population size. Thus, the stochastic nature of biological processes involved in viral replication contributes to increased genetic drift in HIV populations. We propose that appreciation of these effects will allow better understanding of the evolutionary forces acting on HIV in infected patients.     

Vaginal Challenge with an SIV-Based Dual Reporter System Reveals That Infection Can Occur throughout the Upper and Lower Female Reproductive Tract

The majority of new HIV infections occur in women as a result of heterosexual intercourse, overcoming multiple innate barriers to infection within the mucosa. However, the avenues through which infection is established, and the nature of bottlenecks to transmission, have been the source of considerable investigation and contention. Using a high dose of a single round non-replicating SIV-based vector containing a novel dual reporter system, we determined the sites of infection by the inoculum using the rhesus macaque vaginal transmission model. Here we show that the entire female reproductive tract (FRT), including the vagina, ecto- and endocervix, along with ovaries and local draining lymph nodes can contain transduced cells only 48 hours after inoculation. The distribution of infection shows that virions quickly disseminate after exposure and can access target cells throughout the FRT, with an apparent preference for infection in squamous vaginal and ectocervical mucosa. JRFL enveloped virions infect diverse CD4 expressing cell types, with T cells resident throughout the FRT representing the primary target. These findings establish a new perspective that the entire FRT is susceptible and virus can reach as far as the ovary and local draining lymph nodes. Based on these findings, it is essential that protective mechanisms for prevention of HIV acquisition must be present at protective levels throughout the entire FRT to provide complete protection.     

High level of coreceptor-independent HIV transfer induced by contacts between primary CD4 T cells

Cell-to-cell virus transmission is one of the most efficient mechanisms of human immunodeficiency virus (HIV) spread, requires CD4 and coreceptor expression in target cells, and may also lead to syncytium formation and cell death. Here, we show that in addition to this classical coreceptor-mediated transmission, the contact between HIV-producing cells and primary CD4 T cells lacking the appropriate coreceptor induced the uptake of HIV particles by target cells in the absence of membrane fusion or productive HIV replication. HIV uptake by CD4 T cells required cellular contacts mediated by the binding of gp120 to CD4 and intact actin cytoskeleton. HIV antigens taken up by CD4 T cells were rapidly endocytosed to trypsin-resistant compartments inducing a partial disappearance of CD4 molecules from the cell surface. Once the cellular contact was stopped, captured HIV were released as infectious particles. Electron microscopy revealed that HIV particles attached to the surface of target cells and accumulated in large (0.5-1.0 microm) intracellular vesicles containing 1-14 virions, without any evidence for massive clathrin-mediated HIV endocytosis. The capture of HIV particles into trypsin-resistant compartments required the availability of the gp120 binding site of CD4 but was independent of the intracytoplasmic tail of CD4. In conclusion, we describe a novel mechanism of HIV transmission, activated by the contact of infected and uninfected primary CD4 T cells, by which HIV could exploit CD4 T cells lacking the appropriate coreceptor as an itinerant virus reservoir.     

HIV-1 utilizes the CXCR4 chemokine receptor to infect multipotent hematopoietic stem and progenitor cells

HIV infection is characterized by gradual immune system collapse and hematopoietic dysfunction. We recently showed that HIV enters multipotent hematopoietic progenitor cells and establishes both active cytotoxic and latent infections that can be reactivated by myeloid differentiation. However, whether these multipotent progenitors include long-lived hematopoietic stem cells (HSCs) that could establish viral reservoirs for the life of the infected person remains unknown. Here we provide direct evidence that HIV targets long-lived HSCs and show that infected HSCs yield stable, multilineage engraftment in a xenograft model. Furthermore, we establish that the capacity to use the chemokine receptor CXCR4 for entry determines whether a virus will enter multipotent versus differentiated progenitor cells. Because HSCs live for the life span of the infected person and are crucial for hematopoietic health, these data may explain the poor prognosis associated with CXCR4-tropic HIV infection and suggest HSCs as long-lived cellular reservoirs of latent HIV.     

Analytic results for quantifying HIV infectivity

In a separate paper, we developed a mathematical model describing HIV infection and used it to suggest experiments for quantifying characteristic viral parameters. In this paper we generalize the model to any well-mixed assay system. We also present complete and rigorous derivations of fundamental results needed for the design and analysis of HIV infectivity assays. The model is applicable to infectious agents with multiple receptors for their target cell (e.g. HIV, Epstein-Barr virus and Plasmodium), and to blockers (both reversible and irreversible), as long as blocker and target cells are the same diffusion compartment.     

Coreceptor Switching in HIV-1 Subtype B and Subtype C

We use a mathematical model to determine the factors affecting the delayed or rare coreceptor switch in HIV-1 subtype C infected individuals. The model takes into account the two main target cells for the CXCR4-tropic and CCR5-tropic virus and includes the the lytic and non-lytic immune responses. Computer-based simulations and a sensitivity analysis of the model predict that a persistent immune response suppresses the CXCR4-tropic virus to low levels and hence preventing a phenotypic switch. However, not only should the immune response be persistent, but it should have an efficient lytic immune response rather that an efficient non-lytic response. In addition, we also find that the availability of macrophage cells and enhanced viral kinetics are also crucial for the dominance of the R5 strain. We suggest that an altered host environment probably as a result of immune activation may explain the difference in coreceptor switching kinetics between HIV-1 subtype B and subtype C individuals.     

Activated CD34-derived Langerhans cells mediate transinfection with human immunodeficiency virus

Langerhans cells (LCs) are a subset of dendritic cells (DCs) that reside within epidermal and mucosal tissue. Because of their location, LCs are potentially the first cells to encounter human immunodeficiency virus (HIV) during sexual transmission. We report that LCs purified from CD34(+)-derived DCs can facilitate the transinfection of target cells but only after activation. Virions were observed in an intracellular compartment that contains several tetraspanins, in addition to the unique LC markers langerin and CD1a. This reveals that the trafficking of HIV within LCs is reminiscent of that which occurs in mature monocyte-derived DCs and that it varies with the activation state of the cell. The observation that activated LCs can mediate transinfection suggests a potential role for these cells in the known increase in HIV transmission associated with sexually transmitted infections that would cause inflammation of the genital lining.     

Immune responses and the emergence of drug-resistant virus strains in vivo

The treatment of viral infections using antiviral drugs has had a significant public health benefit in the setting of human immunodeficiency virus (HIV) infection, and newly developed drugs offer potential benefits in the management of other viral infections, including acute self-limiting infections such as influenza and picornaviruses (including the rhinoviruses that are responsible for a large proportion of 'common colds'). A serious concern with such treatments is that they may lead to the selection of drug-resistant strains. This has been a significant problem in the case of HIV infection. Existing mathematical-modelling studies of drug resistance have focused on the interactions between virus, target cells and infected cells, ignoring the impact of immune responses. Here, we present a model that explores the role of immune responses in the rise of drug-resistant mutants in vivo. We find that drug resistance is unlikely to be a problem if immune responses are maintained above a threshold level during therapy. Alternatively, if immune responses decline at a fast rate and fall below a threshold level during treatment (indicating impaired immunity), the rise of drug-resistant mutants is more likely. This indicates an important difference between HIV, which impairs immunity and for which immune responses have been observed to vanish during treatment, and viral infections such as influenza and rhinoviruses, for which such immune impairment is not present. Drug resistance is much more likely to be a problem in HIV than in acute and self-limiting infections.     

In vitro infection of natural killer cells with different human immunodeficiency virus type 1 isolates.

Natural killer (NK) cells are a discrete subset of leukocytes, distinct from T and B lymphocytes. NK cells mediate spontaneous non-MHC-restricted killing of a wide variety of target cells without prior sensitization and appear to be involved in initial protection against certain viral infections. Depressed NK cell-mediated cytotoxicity, one of the many immunological defects observed in AIDS patients, may contribute to secondary virus infections. Here we report that clonal and purified polyclonal populations of NK cells, which expressed neither surface CD4 nor CD4 mRNA, were susceptible to infection with various isolates of human immunodeficiency virus type 1 (HIV-1). Viral replication was demonstrated by detection of p24 antigen intracellularly and in culture supernatants, by the presence of HIV DNA within infected cells, and by the ability of supernatants derived from HIV-infected NK cells to infect peripheral blood mononuclear cells or CD4+ cell lines. Infection of NK cells was not blocked by anti-CD4 or anti-Fc gamma RIII monoclonal antibodies. NK cells from HIV-infected and uninfected cultures were similar in their ability to lyse three different target cells. Considerable numbers of cells died in HIV-infected NK cell cultures. These results suggest that loss of NK cells in AIDS patients is a direct effect of HIV infection but that reduced NK cell function involves another mechanism. The possibility that NK cells serve as a potential reservoir for HIV-1 must be considered.     

The dual role of CD4 T helper cells in the infection dynamics of HIV and their importance for vaccination

Given the role of the CD4 T helper cells in the development of memory CTL precursors, it seems beneficial to boost the CD4 T helper response in the context of vaccination against the human immunodeficiency virus (HIV). However, CD4 T cells are also the preferred targets of infection by HIV. Here, we address the question as to whether it is advantageous to stimulate the CD4 T helper cell response, as this will increase the pool of potential target cells of infection. To do so we formulated a mathematical model describing the interactions between virus-infected cells, susceptible cells, HIV-specific CD4 helper T cells, and CTL precursor (CTLp) and effector cells (CTLe). The effect of increased initial CD4 helper and CTLp numbers on the outcome of infection, as well as the effect on viral set point of increased CD4 T helper growth rate, CTL responsiveness and the rate at which CTLp and CTLe are produced were studied. We found that only when the virus has a low basic reproductive number does the number of CTLp and CD4 T helper cells at the moment of infection influence the outcome of infection. In this situation, high initial T helper and CTL numbers can switch the outcome from full-blown infection to virus control. However, this holds for virus with infectivity in a limited range, and current estimates of virus infectivity suggest that it is higher. In that case, only a vaccination protocol that increases CTL responsiveness, ideally in combination with the rate of production of CD4 T helper cells, may offer a solution as it can reduce the viral set point considerably. If brought under a certain level, the viral population might be unable to replicate any further. However, changing these parameters of the immune response is only beneficial when infection is controlled by CTL in the long term. When a CD4 lymphoproliferative response is mounted but the CTL response is not maintained, increasing the CD4 T helper growth rate is deleterious.