Diffuse large B-cell lymphoma (DLBCL) is more prevalent and more often fatal in HIV-infected patients and SIV-infected monkeys compared to immune-competent individuals. Molecular, biological, and immunological data indicate that virus-associated lymphomagenesis is similar in both infected hosts. To find genes specifically overexpressed in HIV/SIV-associated and non-HIV/SIV-associated DLBCL we compared gene expression profiles of HIV/SIV-related and non-HIV-related lymphomas using subtractive hybridization and Northern blot analysis. Our experimental approach allowed us to detect two genes (a-myb and pub) upregulated solely in HIV/SIV-associated DLBCLs potentially involved in virus-specific lymphomagenesis in human and monkey. Downregulation of the pub gene was observed in all non-HIV-associated lymphomas investigated. In addition, we have found genes upregulated in both non-HIV- and HIV-associated lymphomas. Among those were genes both with known (set, ND4, SMG-1) and unknown functions. In summary, we have demonstrated that simultaneous transcriptional upregulation of at least two genes (a-myb and pub) was specific for AIDS-associated lymphomas.
non-Hodgkin's lymphoma; diffuse large B-cell lymphoma; HIV/SIV-associated lymphomas; spontaneous; differentially expressed genes; subtractive hybridization
Immune surveillance is a dynamic process that involves an intact immune system to identify and protect the host against tumor development. The increased understanding of the genetics, infections and hematological malignancies in congenital immune deficiency states supports the concept that impaired T cells and Natural-killer/T cells leads to B-cell lymphoma. Furthermore, severe combined immunodeficient mice are prone to spontaneous tumor development and therefore serve as experimental models. Here we discuss the acquired conditions and mechanisms involved in dysregulation of the immune system that lead to lymphoma. Preemptive strategies to improve immune regulation and response and restore a competent immune system may lead to a decrease in lymphoid malignancies.
Lymphoma; immune surveillance; immune deficiency
The persistence of a reservoir of transcriptionally competent but latent virus in the presence of antiviral regimens presents the main impediment to a curative therapy against HIV. Therefore it is critical to understand the molecular mechanisms, which lead to the establishment and maintenance of HIV latency, and which contribute to the reversal of this process and mediate HIV transcriptional activation in response to T cell activation signals. Here I discuss features of the nucleosomal landscape of the HIV promoter or 5'LTR in controlling HIV transcription. I emphasize on the emerging understanding of the role of the ATP dependent SWI/SNF chromatin remodelling complexes in modulating the chromatin architecture at the HIV LTR and how this leads to a tight regulation of LTR transcription.
BAF; HIV-1; SWI/SNF; chromatin structure; nucleosome positioning; transcription factors
B cell non Hodgkin lymphomas account for the majority of lymphomas in Uganda. The commonest is endemic Burkitt lymphoma, followed by diffuse large-B-cell lymphoma (DLBCL). There has been an increase in incidence of malignant lymphoma since the onset of the HIV/AIDS pandemic. However, the possible linkages of HHV8 and EBV to the condition of impaired immunity present in AIDS are still not yet very clearly understood.
1. To describe the prevalence of Epstein-Barr virus, Human Herpes virus 8 and Human Immunodeficiency Virus-1 in B cell non Hodgkin lymphoma biopsy specimens in Kampala, Uganda.
2. To describe the histopathology of non Hodgkin lymphoma by HIV serology test result in Kampala, Uganda
Tumour biopsies specimens from 119 patients with B cell non Hodgkin lymphoma were classified according to the WHO classification. Immunohistochemistry was used for detection of HHV8 and in situ hybridization with Epstein Barr virus encoded RNA (EBER) for EBV. Real time and nested PCR were used for the detection of HIV.
The patients from whom the 1991-2000 NHL biopsies had been taken did not have HIV serology results therefore 145 patients biopsies where serology results were available were used to describe the association of HIV with non Hodgkin lymphoma type during 2008-2009.
In this study, the majority (92%) of the Burkitt lymphomas and only 34.8% of the diffuse large B cell lymphomas were EBV positive. None of the precursor B lymphoblastic lymphomas or the mantle cell lymphomas showed EBV integration in the lymphoma cells.
None of the Burkitt lymphoma biopsies had HIV by PCR. Of the 121 non Hodgkin B cell lymphoma patients with HIV test results, 19% had HIV. However, only 1(0.04%) case of Burkitt lymphoma had HIV. All the tumours were HHV8 negative.
The majority of the Burkitt lymphomas and two fifths of the diffuse large B cell lymphomas had EBV. All the tumours were HHV8 negative. Generally, the relationship of NHL and HIV was weaker than what has been reported from the developed countries. We discuss the role of these viruses in lymphomagenesis in light of current knowledge.
We use mathematical models to determine possible mechanisms contributing to the evolution and rise of virulent CXCR4-tropic HIV in vivo. The models predict that the ability of the virus to specialize on a given target cell type depends on the exact fitness landscape of the viral mutants. Because this fitness landscape varies between people, this may explain why the evolution of fully CXCR4-tropic strains only occurs in about 50% of infected patients. Assuming that CXCR4-tropic HIV may evolve, we investigate the effect of different immune responses on the rise of such virulent strains. If we assume that CXCR4-tropic HIV is more cytopathic than CCR5-tropic virus, virulent CXCR4-tropic mutants remain suppressed at low levels both in the absence of an immune response, and in the presence of responses that act on the virus before integration into the host genome. On the other hand, this difference in cytopathogenicity is reduced by the presence of immune responses acting on infected cells, allowing CXCR4-tropic HIV to coexist with the CCR5-tropic virus. These results may help to interpret experimental data and are discussed with reference to the literature.
The HIV epidemic has challenged our previous understanding of endemic Burkitt's lymphoma. Despite the strong association of Burkitt's lymphoma and HIV infection in the Developed world, and against previous postulations that the cancer is due to immunosupression among African children, the HIV epidemic in the Malaria belt has not been associated with a corresponding increase in incidence of childhood Burkitt's lymphoma. Even outside the context of HIV infection, there is substantial evidence for a strong but skewed immune response towards a TH2 response in genesis of Burkitt lymphoma.
Presentation of the hypothesis
Rather than a global and/or profound immunosupression, the final common pathway in genesis of Burkitt's lymphoma is the dysregulation of the immune response towards a TH2 response dominated by B-lymphocytes, and the concomitant suppression of the TH1 cell-mediated immune surveillance, driven by various viral/parasitic/bacterial infections.
Testing the hypothesis
Case control studies comparing TH2 and TH1 immune responses in Burkitt lymphoma of different etiological types (sporadic, HIV-related, endemic and post-transplant) to demonstrate significant dominance of TH2 immune response in presence of poor CMI response as a common factor. Immunological profiling to evaluate differences between immune states that are associated (such as recurrent Malaria infection) and those that are not associated (such as severe protein-energy malnutrition) with Burkitt lymphoma. Prospective cohorts profiling chronology of immunological events leading to Burkitt lymphoma in children with EBV infection.
Implications of the hypothesis
The dysregulation of the immune response may be the missing link in our understanding of Burkitt lymphomagenesis. This will provide possibilities for determination of risk and for control of development of malignancy in individuals/populations exposed to the relevant infections.
Epstein Barr virus (EBV) causes lymphomas in immune competent and, at increased frequencies, in immune compromised patients. In the presence of an intact immune system, EBV associated lymphomas express in most cases only three or fewer EBV antigens at the protein level, always including the nuclear antigen 1 of EBV (EBNA1). EBNA1 is a prominent target for EBV specific CD4+ T cell and humoral immune responses in healthy EBV carriers. Here we demonstrate that patients with EBV associated lymphomas, primarily Hodgkin's lymphoma, lack detectable EBNA1 specific CD4+ T cell responses and have slightly altered EBNA1 specific antibody titers at diagnosis. In contrast, the majority of EBV negative lymphoma patients had detectable IFNγ expression and proliferation by CD4+ T cells in response to EBNA1, and carry EBNA1 specific immunoglobulins at levels similar to healthy virus carriers. Other EBV antigens, which were not present in the tumors, were recognized in less EBV positive, than negative lymphoma patients, but detectable responses reached similar CD8+ T cell frequencies in both cohorts. Patients with EBV positive and negative lymphomas did not differ in T cell responses in influenza specific CD4+ T cell proliferation and in antibody titers against tetanus toxoid. These data suggest a selective loss of EBNA1 specific immune control in EBV associated lymphoma patients, which should be targeted for immunotherapy of these malignancies.
EBNA1; CD4+ T cells; Hodgkin's lymphoma
Failure on Highly Active Anti-Retroviral Treatment is often accompanied with development of antiviral resistance to one or more drugs included in the treatment. In general, the virus is more likely to develop resistance to drugs with a lower genetic barrier. Previously, we developed a method to reverse engineer, from clinical sequence data, a fitness landscape experienced by HIV-1 under nelfinavir (NFV) treatment. By simulation of evolution over this landscape, the individualized genetic barrier to NFV resistance may be estimated for an isolate.
We investigated the association of estimated genetic barrier with risk of development of NFV resistance at virological failure, in 201 patients that were predicted fully susceptible to NFV at baseline, and found that a higher estimated genetic barrier was indeed associated with lower odds for development of resistance at failure (OR 0.62 (0.45 - 0.94), per additional mutation needed, p = .02).
Thus, variation in individualized genetic barrier to NFV resistance may impact effective treatment options available after treatment failure. If similar results apply for other drugs, then estimated genetic barrier may be a new clinical tool for choice of treatment regimen, which allows consideration of available treatment options after virological failure.
Enteroviruses elicit protective mucosal immune responses that could be harnessed as part of a strategy to prevent sexual transmission of the human immunodeficiency virus-1 (HIV-1). We report the construction of replication competent recombinant vectors of coxsackievirus B3 (CVB3) that express one or more portions of the HIV-1 Gag protein. Vectors containing the capsid domain of Gag were initially genetically unstable with protein expression lost after brief passage in tissue culture. Codon modification to increase the G/C content of the HIV-1 capsid sequence resulted in enhanced genetic stability of CVB3 vectors during in vitro passage. Cells infected with a vector expressing the matrix (MA) subunit of the HIV-1 Gag protein were susceptible to lysis by CD8 T cell clones specific for the SL9 epitope found within MA. These studies suggest that CVB3 vectors may be useful as vaccine vector candidates, if hurdles in class I antigen presentation and stability can be overcome.
HIV-1; Vaccine; Coxsackievirus B3
Tomasz, Alexander (The Rockefeller Institute, New York, N.Y.), and Samuel M. Beiser. Relationship between the competence antigen and the competence-activator substance in pneumococci. J. Bacteriol. 90:1226–1232. 1965.—Antisera prepared against pneumococci in their competent phase inhibit deoxyribonuoleic acid (DNA)-mediated genetic transformation as well as binding of radioactive DNA by the cells. The same sera do not inhibit transformation of competent Haemophilus influenzae and Bacillus subtilis cells, but transformation of a Streptococcus strain genetically related to pneumococci is inhibited. The kinetics of immune inhibition of transformation resembles the inactivation of bacteriophage by phage-neutralizing antisera. The appearance of the competence antigen on the surface of pneumococci can be induced by the competence-activator substance. Antisera prepared against competent pneumococci can also inhibit the conversion of incompetent cells to competence by the competence-activator substance. The possibility is considered that part of the new antigenic determinant appearing on the cell surface during competence may be the activator itself.
Viral quasispecies can be regarded as a swarm of genetically related mutants. A common approach employed to describe viral quasispecies is by means of the quasispecies equation (QE). However, a main criticism of QE is its lack of frequency-dependent selection. This can be overcome by an alternative formulation for the evolutionary dynamics: the replicator-mutator equation (RME). In turn, a problem with the RME is how to quantify the interaction coefficients between viral variants. Here, this is addressed by adopting an ecological perspective and resorting to the niche theory of competing communities, which assumes that the utilization of resources primarily determines ecological segregation between competing individuals (the different viral variants that constitute the quasispecies). This provides a theoretical framework to estimate quantitatively the fitness landscape.
Using this novel combination of RME plus the ecological concept of niche overlapping for describing a quasispecies we explore the population distributions of viral variants that emerge, as well as the corresponding dynamics. We observe that the population distribution requires very long transients both to A) reach equilibrium and B) to show a clear dominating master sequence. Based on different independent and recent experimental evidence, we find that when some cooperation or facilitation between variants is included in appropriate doses we can solve both A) and B). We show that a useful quantity to calibrate the degree of cooperation is the Shannon entropy.
In order to get a typical quasispecies profile, at least within the considered mathematical approach, it seems that pure competition is not enough. Some dose of cooperation among viral variants is needed. This has several biological implications that might contribute to shed light on the mechanisms operating in quasispecies dynamics and to understand the quasispecies as a whole entity.
Experimental populations of Escherichia coli have evolved for 20,000 generations in a uniform environment. Their rate of improvement, as measured in competitions with the ancestor in that environment, has declined substantially over this period. This deceleration has been interpreted as the bacteria approaching a peak or plateau in a fitness landscape. Alternatively, this deceleration might be caused by non-transitive competitive interactions, in particular such that the measured advantage of later genotypes relative to earlier ones would be greater if they competed directly.
To distinguish these two hypotheses, we performed a large set of competitions using one of the evolved lines. Twenty-one samples obtained at 1,000-generation intervals each competed against five genetically marked clones isolated at 5,000-generation intervals, with three-fold replication. The pattern of relative fitness values for these 315 pairwise competitions was compared with expectations under transitive and non-transitive models, the latter structured to produce the observed deceleration in fitness relative to the ancestor. In general, the relative fitness of later and earlier generations measured by direct competition agrees well with the fitness inferred from separately competing each against the ancestor. These data thus support the transitive model.
Non-transitive competitive interactions were not a major feature of evolution in this population. Instead, the pronounced deceleration in its rate of fitness improvement indicates that the population early on incorporated most of those mutations that provided the greatest gains, and subsequently relied on beneficial mutations that were fewer in number, smaller in effect, or both.
Although the high incidence of EBV-associated diffuse large cell lymphomas (DLCL) in HIV-1 infection is believed to be related to loss of immune control due to HIV-induced immune deficiency, it has been claimed that cytotoxic T lymphocyte (CTL) responses to EBV are longer lasting in HIV-1-infected persons than CTL directed against HIV-1 itself. We approached this apparent paradox by performing the first longitudinal study into the kinetics of EBV and HIV-specific CTL responses in HIV-infected patients progressing either to AIDS with non-Hodgkin's lymphoma (NHL) or AIDS with opportunistic infection (OI). Multiple samples were tested from HIV-1 seroconversion to AIDS-diagnosis. Four out of six patients that were either long-term asymptomatic or progressing to OI showed declining HIV-1 CTL precursor (CTLp) frequencies whereas EBV-CTLp remained stable, suggestive for HIV-1-specific immune exhaustion. In two patients rapidly progressing to AIDS-OI, a parallel decline of HIV-1- and EBV-CTL responses was seen, indicative for total collapse of cellular immunity. In all these six patients EBV-load remained low. However, in four out of five patients that progressed to DLCL, EBV-load was high and increasing several months preceding the NHL. In all five patients, EBV-CTLp decreased before the emergence of the NHL. Thus, our data show that in HIV-1 infection loss of HIV-1-specific T cell immunity is not necessarily paralleled by loss of EBV-specific T cell responses. The occurrence of AIDS-related DLCL is preceded by decreasing EBV-CTLp and increasing EBV load. Failing EBV-control might therefore be an important step in the pathogenesis of AIDS-related DLCL.
Summary: Globally, the number of immunosuppressed people increases each year, with the human immunodeficiency virus (HIV) pandemic continuing to spread unabated in many parts of the world. Immunosuppression may also occur in malnourished persons, patients undergoing chemotherapy for malignancy, and those receiving immunosuppressive therapy. Components of the immune system can be functionally or genetically abnormal as a result of acquired (e.g., caused by HIV infection, lymphoma, or high-dose steroids or other immunosuppressive medications) or congenital illnesses, with more than 120 congenital immunodeficiencies described to date that either affect humoral immunity or compromise T-cell function. All individuals affected by immunosuppression are at risk of infection by opportunistic parasites (such as the microsporidia) as well as those more commonly associated with gastrointestinal disease (such as Giardia). The outcome of infection by enteric protozoan parasites is dependent on absolute CD4+ cell counts, with lower counts being associated with more severe disease, more atypical disease, and a greater risk of disseminated disease. This review summarizes our current state of knowledge on the significance of enteric parasitic protozoa as a cause of disease in immunosuppressed persons and also provides guidance on recent advances in diagnosis and therapy for the control of these important parasites.
Current life history theory suggests that the allocation of energetic resources between competing physiological needs should be dictated by an individual’s longevity and pace of life. One key physiological pathway likely to contribute to the partitioning of resources is the vertebrate stress response. By increasing circulating glucocorticoids the stress response can exert a suite of physiological effects, such as altering immune function. We investigated the effects of stress physiology on individual immunity, reproduction and oxidative stress, across an urban landscape. We sampled populations in and around St. George, Utah, examining corticosterone in response to restraint stress, two innate immune measures, reproductive output, and the presence of both reactive oxygen metabolites and antioxidant binding capacity, in populations of common side-blotched lizards (Uta stansburiana) experiencing variable levels of environmental stress. Additionally, using capture-mark-recapture techniques, we examined the relationships between these physiological parameters and population-level differences. Our results reveal elevated physiological stress corresponds with suppressed immunity and increased oxidative stress. Interestingly, urban populations experiencing the most physiological stress also exhibited greater reproductive output and decreased survival relative to rural populations experiencing less physiological stress, demonstrating a tradeoff between reproduction and life maintenance processes. Our results suggest that environmental stress may augment life history strategy in this fast-paced species, and that shifts in life history strategy can in turn affect the population at large. Finally, the urban environment poses definite challenges for organisms, and while it appears that side-blotched lizards are adjusting physiologically, it is unknown what fitness costs these physiological adjustments accrue.
Although fitness landscapes are central to evolutionary theory, so far no biologically realistic examples for large-scale fitness landscapes have been described. Most currently available biological examples are restricted to very few loci or alleles and therefore do not capture the high dimensionality characteristic of real fitness landscapes. Here we analyze large-scale fitness landscapes that are based on predictive models for in vitro replicative fitness of HIV-1. We find that these landscapes are characterized by large correlation lengths, considerable neutrality, and high ruggedness and that these properties depend only weakly on whether fitness is measured in the absence or presence of different antiretrovirals. Accordingly, adaptive processes on these landscapes depend sensitively on the initial conditions. While the relative extent to which mutations affect fitness on their own (main effects) or in combination with other mutations (epistasis) is a strong determinant of these properties, the fitness landscape of HIV-1 is considerably less rugged, less neutral, and more correlated than expected from the distribution of main effects and epistatic interactions alone. Overall this study confirms theoretical conjectures about the complexity of biological fitness landscapes and the importance of the high dimensionality of the genetic space in which adaptation takes place.
Evolutionary adaptation can be understood as populations moving uphill on landscapes, in which height corresponds to evolutionary fitness. Although such fitness landscapes are central to evolutionary theory, there is currently a lack of biologically realistic examples. Here we analyze large-scale fitness landscapes derived from in vitro fitness measurements of HIV-1. We find that these landscapes are very rugged and that, accordingly, adaptive processes on these landscapes depend sensitively on the initial conditions. Moreover, the landscapes contain large networks along which fitness changes only minimally. While the relative extent to which mutations affect fitness on their own or in combination with other mutations is a strong determinant of these properties, the fitness landscape of HIV-1 is considerably less rugged than expected from the individual and pair-wise effects of mutations. Overall this study confirms theoretical conjectures about the complexity of biological fitness landscapes and the importance of the high dimensionality of the genetic space in which adaptation takes place.
The complex hide-and-seek game between HIV-1 and the host immune system has impaired the development of an efficient vaccine. In addition, the high variability of the virus impedes the long-term control of viral replication by small antiviral drugs. For more than 20 years, phage display technology has been intensively used in the field of HIV-1 to explore the epitope landscape recognized by monoclonal and polyclonal HIV-1-specific antibodies, thereby providing precious data about immunodominant and neutralizing epitopes. In parallel, biopanning experiments with various combinatorial or antibody fragment libraries were conducted on viral targets as well as host receptors to identify HIV-1 inhibitors. Besides these applications, phage display technology has been applied to characterize the enzymatic specificity of the HIV-1 protease. Phage particles also represent valuable alternative carriers displaying various HIV-1 antigens to the immune system and eliciting antiviral responses. This review presents and summarizes the different studies conducted with regard to the nature of phage libraries, target display mode and biopanning procedures.
phage display; HIV-1; epitope mapping; mimotopes; HIV-1 inhibitor; HIV-1 vaccine; gp120; gp41; CXCR4; CCR5
The connection between random environments and genetic and phenotypic variability has been a major focus in the population genetic literature. By providing differential access to the underlying genetic information, epigenetic variation could play an important role in the interaction between environmental and phenotypic variation. Using simulation, we model epigenetic plasticity during development by investigating the dynamics of genetic regulators of the epigenetic machinery that change the variance of the phenotype, while having no effect on the phenotype's mean. Previous studies have found that increased phenotypic variance is selected for if the environment is fluctuating. Here, we find that when a variance-increasing allele achieves a sufficiently high frequency, it can be out-competed by a variance-reducing allele, with the consequence that the population evolves to an equilibrium phenotypic variability. This equilibrium is shown to be robust to different initial conditions, but to depend heavily on parameters of the model, such as the mutation rate, the fitness landscape and the nature of the environmental fluctuation. Indeed, if there is no mutation at the genes controlling the variance of the phenotype, reduction of this variance is favoured.
epigenetics; epigenetic variation; phenotypic variation; fluctuating environments; evolution
Epstein-Barr virus (EBV) persistently infects more than 90% of the human population and is etiologically linked to several B cell malignancies, including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B cell lymphoma (DLBCL). Despite its growth transforming properties, most immune-competent individuals control EBV infection throughout their lives. EBV encodes various oncogenes, and of the 6 latency-associated EBV-encoded nuclear antigens, only EBNA3B is completely dispensable for B cell transformation in vitro. Here, we report that infection with EBV lacking EBNA3B leads to aggressive, immune-evading monomorphic DLBCL-like tumors in NOD/SCID/γc–/– mice with reconstituted human immune system components. Infection with EBNA3B-knockout EBV (EBNA3BKO) induced expansion of EBV-specific T cells that failed to infiltrate the tumors. EBNA3BKO-infected B cells expanded more rapidly and secreted less T cell–chemoattractant CXCL10, reducing T cell recruitment in vitro and T cell–mediated killing in vivo. B cell lines from 2 EBV-positive human lymphomas encoding truncated EBNA3B exhibited gene expression profiles and phenotypic characteristics similar to those of tumor-derived lines from the humanized mice, including reduced CXCL10 secretion. Screening EBV-positive DLBCL, HL, and BL human samples identified additional EBNA3B mutations. Thus, EBNA3B is a virus-encoded tumor suppressor whose inactivation promotes immune evasion and virus-driven lymphomagenesis.
The development of an HIV/AIDS vaccine has proven to be elusive. Because human vaccine trials have not yet demonstrated efficacy, new vaccine strategies are needed for the HIV vaccine pipeline. We have been developing a new HIV vaccine platform using a live enterovirus, coxsackievirus B4 (CVB4) vector. Enteroviruses are ideal candidates for development as a vaccine vector for oral delivery, because these viruses normally enter the body via the oral route and survive the acidic environment of the stomach.
We constructed a live coxsackievirus B4 recombinant, CVB4/p24(733), that expresses seventy-three amino acids of the gag p24 sequence (HXB2) and assessed T cell responses after immunization of mice. The CVB4 recombinant was physically stable, replication-competent, and genetically stable. Oral or intraperitoneal immunization with the recombinant resulted in strong systemic gag p24-specific T cell responses as determined by the IFN-γ ELISPOT assay and by multiparameter flow cytometry. Oral immunization with CVB4/p24(733) resulted in a short-lived, localized infection of the gut without systemic spread. Because coxsackieviruses are ubiquitous in the human population, we also evaluated whether the recombinant was able to induce gag p24-specific T cell responses in mice pre-immunized with the CVB4 vector. We showed that oral immunization with CVB4/p24(733) induced gag p24-specific immune responses in vector-immune mice.
The CVB4/p24(733) recombinant retained the physical and biological characteristics of the parental CVB4 vector. Oral immunization with the CVB4 recombinant was safe and resulted in the induction of systemic HIV-specific T cell responses. Furthermore, pre-existing vector immunity did not preclude the development of gag p24-specific T cell responses. As the search continues for new vaccine strategies, the present study suggests that live CVB4/HIV recombinants are potential new vaccine candidates for HIV.
A molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE) has been genetically configured as a replication-competent vaccine vector for the expression of heterologous viral proteins (N. L. Davis, K. W. Brown, and R. E. Johnston, J. Virol. 70:3781-3787, 1996). The matrix/capsid (MA/CA) coding domain of human immunodeficiency virus type 1 (HIV-1) was cloned into the VEE vector to determine the ability of a VEE vector to stimulate an anti-HIV immune response in mice. The VEE-MA/CA vector replicated rapidly in the cytoplasm of baby hamster kidney (BHK) cells and expressed large quantities of antigenically identifiable MA/CA protein. When injected subcutaneously into BALB/c mice, the vector invaded and replicated in the draining lymphoid tissues, expressing HIV-1 MA/CA at a site of potent immune activity. Anti-MA/CA immunoglobulin G (IgG) and IgA antibodies were present in serum of all immunized mice, and titers increased after a second booster inoculation. IgA antibodies specific for MA/CA were detected in vaginal washes of mice that received two subcutaneous immunizations. Cytotoxic T-lymphocyte responses specific for MA/CA were detected following immunization with the MA/CA-expressing VEE vector. These findings demonstrate the ability of a VEE-based vaccine vector system to stimulate a comprehensive humoral and cellular immune response. The multifaceted nature of this response makes VEE an attractive vaccine for immunization against virus infections such as HIV-1, for which the correlates of protective immunity remain unclear, but may include multiple components of the immune system.
Inference concerning the impact of habitat fragmentation on dispersal and gene flow is a key theme in landscape genetics. Recently, the ability of established approaches to identify reliably the differential effects of landscape structure (e.g. land-cover composition, remnant vegetation configuration and extent) on the mobility of organisms has been questioned. More explicit methods of predicting and testing for such effects must move beyond post hoc explanations for single landscapes and species. Here, we document a process for making a priori predictions, using existing spatial and ecological data and expert opinion, of the effects of landscape structure on genetic structure of multiple species across replicated landscape blocks. We compare the results of two common methods for estimating the influence of landscape structure on effective distance: least-cost path analysis and isolation-by-resistance. We present a series of alternative models of genetic connectivity in the study area, represented by different landscape resistance surfaces for calculating effective distance, and identify appropriate null models. The process is applied to ten species of sympatric woodland-dependant birds. For each species, we rank a priori the expectation of fit of genetic response to the models according to the expected response of birds to loss of structural connectivity and landscape-scale tree-cover. These rankings (our hypotheses) are presented for testing with empirical genetic data in a subsequent contribution. We propose that this replicated landscape, multi-species approach offers a robust method for identifying the likely effects of landscape fragmentation on dispersal.
Today, most wild tigers live in small, isolated Protected Areas within human dominated landscapes in the Indian subcontinent. Future survival of tigers depends on increasing local population size, as well as maintaining connectivity between populations. While significant conservation effort has been invested in increasing tiger population size, few initiatives have focused on landscape-level connectivity and on understanding the effect different landscape elements have on maintaining connectivity. We combined individual-based genetic and landscape ecology approaches to address this issue in six protected areas with varying tiger densities and separation in the Central Indian tiger landscape. We non-invasively sampled 55 tigers from different protected areas within this landscape. Maximum-likelihood and Bayesian genetic assignment tests indicate long-range tiger dispersal (on the order of 650 km) between protected areas. Further geo-spatial analyses revealed that tiger connectivity was affected by landscape elements such as human settlements, road density and host-population tiger density, but not by distance between populations. Our results elucidate the importance of landscape and habitat viability outside and between protected areas and provide a quantitative approach to test functionality of tiger corridors. We suggest future management strategies aim to minimize urban expansion between protected areas to maximize tiger connectivity. Achieving this goal in the context of ongoing urbanization and need to sustain current economic growth exerts enormous pressure on the remaining tiger habitats and emerges as a big challenge to conserve wild tigers in the Indian subcontinent.
Naturally occurring Vif variants that are unable to inhibit the host restriction factor APOBEC3G (A3G) have been isolated from infected individuals. A3G can potentially induce G-to-A hypermutation in these viruses, and hypermutation could contribute to genetic variation in HIV-1 populations through recombination between hypermutant and wild-type genomes. Thus, hypermutation could contribute to the generation of immune escape and drug resistant variants, but the genetic contribution of hypermutation to the viral evolutionary potential is poorly understood. In addition, the mechanisms by which these viruses persist in the host despite the presence of A3G remain unknown.
To address these questions, we generated a replication-competent HIV-1 Vif mutant in which the A3G-binding residues of Vif, Y40RHHY44, were substituted with five alanines. As expected, the mutant was severely defective in an A3G-expressing T cell line and exhibited a significant delay in replication kinetics. Analysis of viral DNA showed the expected high level of G-to-A hypermutation; however, we found substantially reduced levels of G-to-A hypermutation in intracellular viral RNA (cRNA), and the levels of G-to-A mutations in virion RNA (vRNA) were even further reduced. The frequencies of hypermutation in DNA, cRNA, and vRNA were 0.73%, 0.12%, and 0.05% of the nucleotides sequenced, indicating a gradient of hypermutation. Additionally, genomes containing start codon mutations and early termination codons within gag were isolated from the vRNA.
These results suggest that sublethal levels of hypermutation coupled with purifying selection at multiple steps during the early phase of viral replication lead to the packaging of largely unmutated genomes, providing a mechanism by which mutant Vif variants can persist in infected individuals. The persistence of genomes containing mutated gag genes despite this selection pressure indicates that dual infection and complementation can result in the packaging of hypermutated genomes which, through recombination with wild-type genomes, could increase viral genetic variation and contribute to evolution.
We investigated the spatial genetic structure of the tiger meta-population in the Satpura–Maikal landscape of central India using population- and individual-based genetic clustering methods on multilocus genotypic data from 273 individuals. The Satpura–Maikal landscape is classified as a global-priority Tiger Conservation Landscape (TCL) due to its potential for providing sufficient habitat that will allow the long-term persistence of tigers. We found that the tiger meta-population in the Satpura–Maikal landscape has high genetic variation and very low genetic subdivision. Individual-based Bayesian clustering algorithms reveal two highly admixed genetic populations. We attribute this to forest connectivity and high gene flow in this landscape. However, deforestation, road widening, and mining may sever this connectivity, impede gene exchange, and further exacerbate the genetic division of tigers in central India.
Central India; connectivity; non-invasive genetic analysis; Panthera tigris; spatial genetics; tiger