Effective Simian Immunodeficiency Virus-Specific CD8+ T Cells Lack an Easily Detectable,Shared Characteristic

The immune correlates of human/simian immunodeficiency virus control remain elusive. While CD8⁺ T lymphocytes likely play a major role in reducing peak viremia and maintaining viral control in the chronic phase, the relative antiviral efficacy of individual virus-specific effector populations is unknown. Conventional assays measure cytokine secretion of virus-specific CD8⁺ T cells after cognate peptide recognition. Cytokine secretion, however, does not always directly translate into antiviral efficacy. Recently developed suppression assays assess the efficiency of virus-specific CD8⁺ T cells to control viral replication, but these assays often use cell lines or clones. We therefore designed a novel virus production assay to test the ability of freshly ex vivo-sorted simian immunodeficiency virus (SIV)-specific CD8⁺ T cells to suppress viral replication from SIVmac239-infected CD4⁺ T cells. Using this assay, we established an antiviral hierarchy when we compared CD8⁺ T cells specific for 12 different epitopes. Antiviral efficacy was unrelated to the disease status of each animal, the protein from which the tested epitopes were derived, or the major histocompatibility complex (MHC) class I restriction of the tested epitopes. Additionally, there was no correlation with the ability to suppress viral replication and epitope avidity, epitope affinity, CD8⁺ T-cell cytokine multifunctionality, the percentage of central and effector memory cell populations, or the expression of PD-1. The ability of virus-specific CD8⁺ T cells to suppress viral replication therefore cannot be determined using conventional assays. Our results suggest that a single definitive correlate of immune control may not exist; rather, a successful CD8⁺ T-cell response may be comprised of several factors.CD8⁺ T cells may play a critical role in blunting peak viremia and controlling human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. The transient depletion of CD8⁺ cells in SIV-infected macaques results in increased viral replication (26, 31, 51, 70). The emergence of virus-specific CD8⁺ T cells coincides with the reduction of peak viremia (12, 39, 42, 63), and CD8⁺ T-cell pressure selects for escape mutants (6, 9, 13, 28, 29, 38, 60, 61, 85). Furthermore, particular major histocompatibility complex (MHC) class I alleles are overrepresented in SIV- and HIV-infected elite controllers (15, 29, 33, 34, 46, 56, 88).Because it has been difficult to induce broadly neutralizing antibodies (Abs), the AIDS vaccine field is currently focused on developing a vaccine designed to elicit HIV-specific CD8⁺ T cells (8, 52, 53, 82). Investigators have tried to define the immune correlates of HIV control. Neither the magnitude nor the breadth of epitopes recognized by virus-specific CD8⁺ T-cell responses correlates with the control of viral replication (1). The quality of the immune response may, however, contribute to the antiviral efficacy of the effector cells. It has been suggested that the number of cytokines that virus-specific CD8⁺ T cells secrete may correlate with viral control, since HIV-infected nonprogressors appear to maintain CD8⁺ T cells that secrete several cytokines, compared to HIV-infected progressors (11, 27). An increased amount of perforin secretion may also be related to the proliferation of HIV-specific CD8⁺ T cells in HIV-infected nonprogressors (55). While those studies offer insight into the different immune systems of progressors and nonprogressors, they did not address the mechanism of viral control. Previously, we found no association between the ability of SIV-specific CD8⁺ T-cell clones to suppress viral replication in vitro and their ability to secrete gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), or interleukin-2 (IL-2) (18).Evidence suggests that some HIV/SIV proteins may be better vaccine targets than others. CD8⁺ T cells recognize epitopes derived from Gag as early as 2 h postinfection, whereas CD8⁺ T cells specific for epitopes in Env recognize infected cells only at 18 h postinfection (68). Additionally, a previously reported study of HIV-infected individuals showed that an increased breadth of Gag-specific responses was associated with lower viral loads (35, 59, 65, 66). CD8⁺ T-cell responses specific for Env, Rev, Tat, Vif, Vpr, Vpu, and Nef were associated with higher viral loads, with increased breadth of Env in particular being significantly associated with a higher chronic-phase viral set point.None of the many sophisticated methods employed for analyzing the characteristics of HIV- or SIV-specific immune responses clearly demarcate the critical qualities of an effective antiviral response. In an attempt to address these questions, we developed a new assay to measure the antiviral efficacy of individual SIV-specific CD8⁺ T-cell responses sorted directly from fresh peripheral blood mononuclear cells (PBMC). Using MHC class I tetramers specific for the epitope of interest, we sorted freshly isolated virus-specific CD8⁺ T cells and determined their ability to suppress virus production from SIV-infected CD4⁺ T cells. We then looked for a common characteristic of efficacious epitope-specific CD8⁺ T cells using traditional methods.     

Simian Immunodeficiency Virus-Specific CD8+ T Cells Recognize Vpr- and Rev-Derived Epitopes Early after Infection

The kinetics of CD8⁺ T cell epitope presentation contribute to the antiviral efficacy of these cells yet remain poorly defined. Here, we demonstrate presentation of virion-derived Vpr peptide epitopes early after viral penetration and prior to presentation of Vif-derived epitopes, which required de novo Vif synthesis. Two Rev epitopes exhibited differential presentation kinetics, with one Rev epitope presented within 1 h of infection. We also demonstrate that cytolytic activity mirrors the recognition kinetics of infected cells. These studies show for the first time that Vpr- and Rev-specific CD8⁺ T cells recognize and kill simian immunodeficiency virus (SIV)-infected CD4⁺ T cells early after SIV infection.The antiviral activity of AIDS virus-specific CD8⁺ T cells is well documented in both in vivo (1, 4, 21) and in vitro (8, 24, 29) studies. Accordingly, human immunodeficiency virus (HIV) vaccine modalities that focus on engendering antiviral CD8⁺ T cells are being developed (13, 26, 28). Ideally, a CD8⁺ T cell-based vaccine would stimulate responses against epitopes that are presented by major histocompatibility complex class I (MHC-I) molecules early after infection of a target cell. However, successful selection of antigenic sequences for a CD8⁺ T cell-based vaccine has been frustrated in part by an incomplete understanding of the properties of effective CD8⁺ T cell responses (25).     

Evolution of broad host range in retroviruses leads to cell death mediated by highly cytopathic variants

The ability of many retroviruses to cause disease can be correlated to their cytopathic effect (CPE) in tissue culture characterized by an acute period of cell death and viral DNA accumulation. Here, we show that mutants of a subgroup B avian retrovirus (Alpharetrovirus) cause a very dramatic CPE in certain susceptible avian cells that is coincident with elevated levels of apoptosis, as measured by nuclear morphology, and persistent viral DNA accumulation. These mutants also have a broadly extended host range that includes rodent, cat, dog, monkey, and human cells (31). Previously, we have shown that the mutants exhibit diminished resistance to superinfection. The results presented here have important implications for the process of evolution of retroviruses to use distinct cellular receptors.     

Gene-environment interactions in a mutant mouse kindred with native airway constrictor hyperresponsiveness

We mutagenized male BTBR mice with N-ethyl-N-nitrosourea and screened 1315 of their G3 offspring for airway hyperresponsiveness. A phenovariant G3 mouse with exaggerated methacholine bronchoconstrictor response was identified and his progeny bred in a nonspecific-pathogen-free (SPF) facility where sentinels tested positive for minute virus of mice and mouse parvovirus and where softwood bedding was used. The mutant phenotype was inherited through G11 as a single autosomal semidominant mutation with marked gender restriction, with males exhibiting almost full penetrance and very few females phenotypically abnormal. Between G11 and G12, facility infection eradication was undertaken and bedding was changed to hardwood. We could no longer detect airway hyperresponsiveness in more than 37 G12 offspring of 26 hyperresponsive G11 males. Also, we could not identify the mutant phenotype among offspring of hyperresponsive G8–G10 sires rederived into an SPF facility despite 21 attempts. These two observations suggest that both genetic and environmental factors were needed for phenotype expression. We suspect that rederivation into an SPF facility or altered exposure to pathogens or other unidentified substances modified environmental interactions with the mutant allele, and so resulted in disappearance of the hyperresponsive phenotype. Our experience suggests that future searches for genes that confer susceptibility for airway hyperresponsiveness might not be able to identify some genes that confer susceptibility if the searches are performed in SPF facilities. Experimenters are advised to arrange for multigeneration constancy of mouse care in order to clone mutant genes. Indeed, we were not able to map the mutation before losing the phenotype.     

Efficacy of RNA amplification is dependent on sequence characteristics: implications for gene expression profiling using a cDNA microarray

Minute tissue samples or single cells increasingly provide the starting material for gene expression profiling, which often requires RNA amplification. Although much effort has been put into optimizing amplification protocols, the relative abundance of RNA templates in the amplified product is frequently biased. We applied a T7 polymerase-based technique to amplify RNA from two tissues of a cichlid fish and compared expression levels of unamplified and amplified RNA on a cDNA microarray. Amplification bias was generally minor and comprised features that were lost (1.3%) or gained (2.5%) through amplification and features that were scored as regulated before but unregulated after amplification (4.2%) or vice versa (19.5%). We examined 10 sequence-specific properties and found that GC content, folding energy, hairpin length and number, and lengths of poly(A) and poly(T) stretches significantly affected RNA amplification. We conclude that, if RNA amplification is used in gene expression studies, preceding experiments controlling for amplification bias should be performed.     

Large rivers do not always act as species barriers for Lepilemur sp.

Sportive lemurs constitute a highly diverse endemic lemur family (24 species) for which many biogeographic boundaries are not yet clarified. Based on recent phylogeographic models, this study aims to determine the importance of two large rivers (the Antainambalana and Rantanabe) in northeastern Madagascar as species barriers for Lepilemur seali. The Antainambalana River was previously assumed to act as the southern border of its distribution. A total of 1,038 bp of the mtDNA of four individuals stemming from two adjacent inter-river systems south of the Antainambalana River was sequenced and compared to sequences of 22 described Lepilemur species. The phylogenetic reconstruction did not find support for either of the two rivers as species barrier for Lepilemur, as all captured individuals clustered closely with and therefore belonged to L. seali. However, a previously published sequence of an individual from a site south of our study sites belongs to a separate species. The southern boundary of L. seali must therefore be one of two large rivers further south of our study sites. The results suggest that L. seali may possess a relatively large altitudinal range that enabled this species to migrate around the headwaters of the Antainambalana and Rantanabe Rivers. Previous phylogeographic models need to be refined in order to incorporate these findings, and more species-specific altitudinal range data are urgently needed in order to fully understand the biogeographic patterns of lemurs on Madagascar.     

Quantifying past and present connectivity illuminates a rapidly changing landscape for the African elephant

There is widespread concern about impacts of land‐use change on connectivity among animal and plant populations, but those impacts are difficult to quantify. Moreover, lack of knowledge regarding ecosystems before fragmentation may obscure appropriate conservation targets. We use occurrence and population genetic data to contrast connectivity for a long‐lived mega‐herbivore over historical and contemporary time frames. We test whether (i) historical gene flow is predicted by persistent landscape features rather than human settlement, (ii) contemporary connectivity is most affected by human settlement and (iii) recent gene flow estimates show the effects of both factors. We used 16 microsatellite loci to estimate historical and recent gene flow among African elephant (Loxodonta africana) populations in seven protected areas in Tanzania, East Africa. We used historical gene flow (F_ST and G'_ST) to test and optimize models of historical landscape resistance to movement. We inferred contemporary landscape resistance from elephant resource selection, assessed via walking surveys across ~15 400 km² of protected and unprotected lands. We used assignment‐based recent gene flow estimates to optimize and test the contemporary resistance model, and to test a combined historical and contemporary model. We detected striking changes in connectivity. Historical connectivity among elephant populations was strongly influenced by slope but not human settlement, whereas contemporary connectivity was influenced most by human settlement. Recent gene flow was strongly influenced by slope but was also correlated with contemporary resistance. Inferences across multiple timescales can better inform conservation efforts on large and complex landscapes, while mitigating the fundamental problem of shifting baselines in conservation.     

Tertiary Mutations Stabilize CD8+ T Lymphocyte Escape-Associated Compensatory Mutations following Transmission of Simian Immunodeficiency Virus

Compensatory mutations offset fitness defects resulting from CD8⁺ T lymphocyte (CD8_TL)-mediated escape, but their impact on viral evolution following transmission to naive hosts remains unclear. Here, we investigated the reversion kinetics of Gag_181–189CM9 CD8_TL escape-associated compensatory mutations in simian immunodeficiency virus (SIV)-infected macaques. Preexisting compensatory mutations did not result in acute-phase escape of the SIVmac239 CD8TL epitope Gag181-189CM9 and instead required a tertiary mutation for stabilization in the absence of Gag_181–189CM9 escape mutations. Therefore, transmitted compensatory mutations do not necessarily predict rapid CD8_TL escape.