Early Selection in Gag by Protective HLA Alleles Contributes to Reduced HIV-1 Replication Capacity That May Be Largely Compensated for in Chronic Infection

Mutations that allow escape from CD8 T-cell responses are common in HIV-1 and may attenuate pathogenesis by reducing viral fitness. While this has been demonstrated for individual cases, a systematic investigation of the consequence of HLA class I-mediated selection on HIV-1 in vitro replication capacity (RC) has not been undertaken. We examined this question by generating recombinant viruses expressing plasma HIV-1 RNA-derived Gag-Protease sequences from 66 acute/early and 803 chronic untreated subtype B-infected individuals in an NL4-3 background and measuring their RCs using a green fluorescent protein (GFP) reporter CD4 T-cell assay. In acute/early infection, viruses derived from individuals expressing the protective alleles HLA-B*57, -B*5801, and/or -B*13 displayed significantly lower RCs than did viruses from individuals lacking these alleles (P < 0.05). Furthermore, acute/early RC inversely correlated with the presence of HLA-B-associated Gag polymorphisms (R = −0.27; P = 0.03), suggesting a cumulative effect of primary escape mutations on fitness during the first months of infection. At the chronic stage of infection, no strong correlations were observed between RC and protective HLA-B alleles or with the presence of HLA-B-associated polymorphisms restricted by protective alleles despite increased statistical power to detect these associations. However, RC correlated positively with the presence of known compensatory mutations in chronic viruses from B*57-expressing individuals harboring the Gag T242N mutation (n = 50; R = 0.36; P = 0.01), suggesting that the rescue of fitness defects occurred through mutations at secondary sites. Additional mutations in Gag that may modulate the impact of the T242N mutation on RC were identified. A modest inverse correlation was observed between RC and CD4 cell count in chronic infection (R = −0.17; P < 0.0001), suggesting that Gag-Protease RC could increase over the disease course. Notably, this association was stronger for individuals who expressed B*57, B*58, or B*13 (R = −0.27; P = 0.004). Taken together, these data indicate that certain protective HLA alleles contribute to early defects in HIV-1 fitness through the selection of detrimental mutations in Gag; however, these effects wane as compensatory mutations accumulate in chronic infection. The long-term control of HIV-1 in some persons who express protective alleles suggests that early fitness hits may provide lasting benefits.The host immune response elicited by CD8⁺ cytotoxic T lymphocytes (CTLs) is a major contributor to viral control following human immunodeficiency virus type 1 (HIV-1) infection (6, 39), but antiviral pressure exerted by CTLs is diminished by the selection of escape mutations in targeted regions throughout the viral proteome (7, 18, 29, 35, 41, 45, 57). A comprehensive identification of HLA-associated viral polymorphisms has recently been achieved through population-based analyses of HIV-1 sequences and HLA class I types from different cohorts worldwide (3, 8, 13-15, 34, 43, 50, 56, 63). However, despite improved characterization of the sites and pathways of immune escape, effective ways to incorporate these findings into immunogen design remain an area of debate. A better understanding of the impact of escape mutations on viral fitness may provide novel directions for HIV-1 vaccines that are designed to attenuate pathogenesis.The development of innovative vaccine strategies that can overcome the extreme diversity of HIV is a key priority (4). One proposed approach is to target the most conserved T-cell epitopes, which presumably cannot escape from CTL pressure easily due to structural or functional constraints on the viral protein (55). Complementary approaches include the design of polyvalent and/or mosaic immunogens that incorporate commonly observed viral diversity (4, 38) or the specific targeting of vulnerable regions of the viral proteome that do escape but only at a substantial cost to viral replication capacity (RC) (1, 40). A chief target of such vaccine approaches is the major HIV-1 structural protein Gag, which is known to be highly immunogenic and to elicit CTL responses that correlate with the natural control of infection (22, 36, 66). Indeed, several lines of evidence support a relationship between the selection of CTL escape mutations and reduced HIV-1 fitness. These include the reversion of escape mutations following transmission to an HLA-mismatched recipient who cannot target the epitope (19, 24, 41) as well as reduced plasma viral load (pVL) set point following the transmission of certain escape variants from donors who expressed protective HLA alleles (17, 27). Notably, these in vivo observations have been made most often for variations within Gag that are attributed to CTL responses restricted by the protective alleles HLA-B*57 and -B*5801 (17, 19, 27, 41). Most recently, reduced in vitro RCs of clinical isolates and/or engineered strains encoding single or multiple escape mutations in Gag selected in the context of certain protective HLA alleles, including B*57, B*5801, B*27, and B*13, have been demonstrated (9, 10, 42, 53, 59, 62). Despite these efforts, the goal of a T-cell vaccine that targets highly conserved and attenuation-inducing sites is hampered by a lack of knowledge concerning the contribution of most escape mutations to HIV-1 fitness as well as a poor understanding of the relative influence of HLA on the viral RC at different stages of infection.The mutability of HIV-1 permits the generation of progeny viruses encoding compensatory mutations that restore normal protein function and/or viral fitness. Detailed studies have demonstrated that the in vitro RC of escape variants in human and primate immunodeficiency viruses can be enhanced by the addition of secondary mutations outside the targeted epitope (10, 20, 52, 59, 65). Thus, vaccine strategies aimed at attenuating HIV-1 must also consider, among other factors, the frequency, time course, and extent to which compensation might overcome attenuation mediated by CTL-induced escape. Despite its anticipated utility for HIV-1 vaccine design, systematic studies to examine the consequences of naturally occurring CTL escape and compensatory mutations on viral RC have not been undertaken.We have described previously an in vitro recombinant viral assay to examine the impact of Gag-Protease mutations on HIV-1 RC (47, 49). Gag and protease have been included in each virus to minimize the impact of sequence polymorphisms at Gag cleavage sites, which coevolve with changes in protease (5, 37). Using this approach, we have demonstrated that viruses derived from HIV-1 controllers replicated significantly less well than those derived from noncontrollers and that these differences were detectable at both the acute/early (49) and chronic (47) stages. Escape mutations in Gag associated with the protective HLA-B*57 allele, as well as putative compensatory mutations outside known CTL epitopes, contributed to this difference in RC (47). However, substantial variability was observed for viruses from controllers and noncontrollers, indicating that additional factors were likely to be involved. Benefits of this assay include its relatively high-throughput capacity as well as the fact that clinically derived HIV-1 sequences are used in their entirety. Thus, it is possible to examine a large number of “real-world” Gag-Protease sequences, to define an RC value for each one, and to identify sequences within the population of recombinant strains that are responsible for RC differences.Here, we use this recombinant virus approach to examine the contribution of HLA-associated immune pressure on Gag-Protease RC during acute/early (n = 66) and chronic (n = 803) infections in the context of naturally occurring HIV-1 subtype B isolates from untreated individuals. In a recent report (64), we employed this system to examine the Gag-Protease RC in a similar cohort of chronic HIV-1 subtype C-infected individuals. The results of these studies provide important insights into the roles of immune pressure and fitness constraints on HIV-1 evolution that may contribute to the rational design of an effective vaccine.     

A cross-sectional single-centre study on Pompe disease in 42 German patients: Molecular analysis of the GAA gene, manifestation and genotype-phenotype correlations

ABSTRACT: BACKGROUND: Pompe disease (Glycogen storage disease type II, GSD II, acid alpha-glucosidase deficiency, acid maltase deficiency, OMIM # 232300) is an autosomal-recessive lysosomal storage disorder due to a deficiency of acid alpha-glucosidase (GAA, acid maltase, EC 3.2.1.20, Swiss-Prot P10253). Clinical manifestations are dominated by progressive weakness of skeletal muscle throughout the clinical spectrum. In addition, the classic infantile form is characterised by hypertrophic cardiomyopathy. Methods: In a cross-sectional single-centre study we clinically assessed 3 patients with classic infantile Pompe disease and 39 patients with non-classic presentations, measured their acid alpha-glucosidase activities and analysed their GAA genes. Results: Classic infantile patients had nearly absent residual enzyme activities and a typical clinical course with hypertrophic cardiomyopathy until the beginning of therapy. The disease manifestations in non-classic patients were heterogeneous. There was a broad variability in the decline of locomotive and respiratory function. The age of onset ranged from birth to late adulthood and correlated with enzyme activities. Molecular analysis revealed as many as 33 different mutations, 14 of which are novel. All classic infantile patients had two severe mutations. The most common mutation in the non-classic group was c.-32-13T>G. It was associated with a milder course in this subgroup. Conclusion: Disease manifestation strongly correlates with the nature of the GAA mutations, while the variable progression in non-classic Pompe disease is likely to be explained by yet unknown modifying factors. This study provides the first comprehensive dataset on the clinical course and the mutational spectrum of Pompe disease in Germany.     

Wind conditions experienced during the day predict nocturnal restlessness in a migratory songbird

A variety of methods have been used to study the relationship between wind conditions and departure decisions of migrant birds at stopover sites. These methods are either costly or suffer from inaccuracy in determining whether or not an individual has resumed migration. Here we present a novel and simple approach to studying the relationship between wind conditions and departure likelihood. Northern Wheatears Oenanthe oenanthe caught during stopover were temporarily caged to measure their nocturnal migratory restlessness, which is an accurate proxy for their individual departure likelihood. We then related the degree of nocturnal restlessness to wind conditions prevailing at the time of capture. Confirming the general pattern from previous studies of departure, the intensity of nocturnal migratory restlessness, and hence departure likelihood, increased with increasing wind support towards the migratory goal. This suggests that approximating the propensity to depart by measuring nocturnal migratory restlessness is a reliable way to study the effect that wind conditions experienced during stopover has on the departure decision of migrants. Our study also shows that nocturnal migrants possess the ability to use information gathered during the day for their departure decisions at night. Because measuring migratory restlessness is straightforward and inexpensive, our approach is ideally suited to test hypotheses regarding spatio‐temporal variation in wind selectivity in migrating birds.     

Interstitial Fluid Flow and Drug Delivery in Vascularized Tumors: A Computational Model

Interstitial fluid is a solution that bathes and surrounds the human cells and provides them with nutrients and a way of waste removal. It is generally believed that elevated tumor interstitial fluid pressure (IFP) is partly responsible for the poor penetration and distribution of therapeutic agents in solid tumors, but the complex interplay of extravasation, permeabilities, vascular heterogeneities and diffusive and convective drug transport remains poorly understood. Here we consider–with the help of a theoretical model–the tumor IFP, interstitial fluid flow (IFF) and its impact upon drug delivery within tumor depending on biophysical determinants such as vessel network morphology, permeabilities and diffusive vs. convective transport. We developed a vascular tumor growth model, including vessel co-option, regression, and angiogenesis, that we extend here by the interstitium (represented by a porous medium obeying Darcy''s law) and sources (vessels) and sinks (lymphatics) for IFF. With it we compute the spatial variation of the IFP and IFF and determine its correlation with the vascular network morphology and physiological parameters like vessel wall permeability, tissue conductivity, distribution of lymphatics etc. We find that an increased vascular wall conductivity together with a reduction of lymph function leads to increased tumor IFP, but also that the latter does not necessarily imply a decreased extravasation rate: Generally the IF flow rate is positively correlated with the various conductivities in the system. The IFF field is then used to determine the drug distribution after an injection via a convection diffusion reaction equation for intra- and extracellular concentrations with parameters guided by experimental data for the drug Doxorubicin. We observe that the interplay of convective and diffusive drug transport can lead to quite unexpected effects in the presence of a heterogeneous, compartmentalized vasculature. Finally we discuss various strategies to increase drug exposure time of tumor cells.     

Stimulation of the diadinoxanthin cycle by UV-B radiation in the diatom Phaeodactylum tricornutum

In this study we show that the diadinoxanthin cycle in the diatom Phaeodactylum tricornutum is stimulated by mild UV-B radiation. High steady state concentrations of diatoxanthin established during a period of strong actinic illumination with white light (300 mol photons m-2 s-1 PAR) are further increased if weak UV-B (3 mol photons m-2 s-1) is additionally applied. Short term increases in the diatoxanthin concentration caused by UV-B strongly correlate with a stoichiometric decrease in diadinoxanthin. The UV-B dependent increase in diatoxanthin is correlated with a concommitant enhancement of non-photochemical quenching of chlorophyll fluorescence and a decrease in the quantum efficiency of oxygen evolution. This indicates that UV-B induced diatoxanthin functions in thermal energy dissipation. Possible scenarios for a stimulation of the diadinoxanthin cycle by UV-B are discussed.     

Identification of a thiolase gene essential for β-oxidation of the acyl side chain of the steroid compound cholate in Pseudomonas sp. strain Chol1

A method to grow the halophilic archaeon Haloferax volcanii in microtiter plates has been optimized and now allows the parallel generation of very reproducible growth curves. The doubling time in a synthetic medium with glucose is around 6 h. The method was used to optimize glucose and casamino acid concentrations, to clarify carbon source usage and to analyze vitamin dependence. The characterization of osmotolerance revealed that after a lag phase of 24 h, H. volcanii is able to grow at salt concentrations as low as 0.7 M NaCl, much lower than the 1.4 M NaCl described as the lowest concentration until now. The application of oxidative stresses showed that H. volcanii exhibits a reaction to paraquat that is delayed by about 10 h. Surprisingly, only one of two amino acid auxotrophic mutants could be fully supplemented by the addition of the respective amino acid. Analysis of eight sRNA gene deletion mutants exemplified that the method can be applied for bona fide phenotyping of mutant collections. This method for the parallel analysis of many cultures contributes towards making H. volcanii an archaeal model species for functional genomic approaches.