Cytokine and Chemokine Signature in Elite Versus Viremic Controllers Infected with HIV.

HIV long-term nonprogressors (LTNPs) maintaining high CD4(+) T-cell counts without antiretroviral therapy (ART) are divided into elite controllers (ECs) with undetectable and viremic controllers (VCs) with low viral loads. Little is known about the long-term changes of T-cell subsets and inflammation patterns in ECs versus VCs. The aim of the study was to explore the long-term evolution of CD4(+) T-cell levels in LTNPs and to analyze cytokine profiles in ECs versus VCs. Nineteen ECs and 15 VCs were enrolled from the natural virus controller cohort (NaViC). T-cell counts were monitored over years, the mean annual change was calculated, and plasma concentrations of 25 cytokines were evaluated using a multiplex bead array. While absolute numbers of T cells did not differ between ECs and VCs over time, we observed a significant decrease of CD4(+) T-cell percentages in VCs, but not in ECs (median [interquartile range]: ECs: 37% [28-41] vs. VCs: 29% [25-34]; p = .02). ECs had lower levels of macrophage inflammatory protein-1ß (MIP-1ß, p = .003), interferon γ-induced protein-10 (IP-10, p = .03), and monokine induced by interferon-γ (MIG, p = .02). CD4(+) T-cell percentages inversely correlated with MIP 1-ß (r = -0.42, p = .017) and IP-10 (r = -0.77, p < .0001). A subtle decline of CD4(+) T-cell percentages could be observed in VCs, but not in ECs, which was associated with higher plasma levels of proinflammatory cytokines. Hence, even low levels of HIV replication might go along with a progressive decline in CD4(+) T-cell counts in LTNPs.

Continued vigilance - development of an online evaluation tool for assessing preparedness of medical facilities for biological events.

OBJECTIVE: Effective response to biological events necessitates ongoing evaluation of preparedness. This study was a bilateral German-Israeli collaboration aimed at developing an evaluation tool for assessing preparedness of medical facilities for biological events. METHODS: Measurable parameters were identified through a literature review for inclusion in the evaluation tool and disseminated to 228 content experts in two modified Delphi cycles. Focus groups were conducted to identify psychosocial needs of the medical teams. Table-top and functional exercises were implemented to review applicability of the tool. RESULTS: One hundred seventeen experts from Germany and Israel participated in the modified Delphi. Out of 188 parameters that were identified, 183 achieved a consensus of >75% of the content experts. Following comments recommended in the Delphi cycles, and feedback from focus groups and hospital exercises, the final tool consisted of 172 parameters. Median level of importance of each parameter was calculated based on ranking recommended in the Delphi process. Computerized web-based software was developed to calculate scores of preparedness for biological events. CONCLUSION: Ongoing evaluation means, such as the tool developed in the study, can facilitate the need for a valid and reliable mechanism that may be widely adopted and implemented as quality assurance measures. The tool is based on measurable parameters and indicators that can effectively present strengths and weaknesses in managing a response to a public health threat, and accordingly, steps can be implemented to improve readiness. Adoption of such a tool is an important component of assuring public health and effective emergency management.

APOBEC3G/F as one possible driving force for co-receptor switch of the human immunodeficiency virus-1.

Human immunodeficiency virus-1 tropism highly correlates with the amino acid (aa) composition of the third hypervariable region (V3) of gp120. A shift towards more positively charged aa is seen when binding to CXCR4 compared with CCR5 (X4 vs. R5 strains), especially positions 11 and 25 (11/25-rule) predicting X4 viruses in the presence of positively charged residues. At nucleotide levels, negatively or uncharged aa, e.g., aspartic and glutamic acid and glycine, which are encoded by the triplets GAN (guanine-adenosine-any nucleotide) or GGN are found more often in R5 strains. Positively charged aa such as arginine and lysine encoded by AAR or AGR (CGN) (R means A or G) are seen more frequently in X4 strains suggesting our hypothesis that a switch from R5 to X4 strains occurs via a G-to-A mutation. 1527 V3 sequences from three independent data sets of X4 and R5 strains were analysed with respect to their triplet composition. A higher number of G-containing triplets was found in R5 viruses, whereas X4 strains displayed a higher content of A-comprising triplets. These findings also support our hypothesis that G-to-A mutations are leading to the co-receptor switch from R5 to X4 strains. Causative agents for G-to-A mutations are the deaminases APOBEC3F and APOBEC3G. We therefore hypothesize that these proteins are one driving force facilitating the appearance of X4 variants. G-to-A mutations can lead to a switch from negatively to positively charged aa and a respective alteration of the net charge of gp120 resulting in a change of co-receptor usage.

Double-stranded RNA analog poly(I:C) inhibits human immunodeficiency virus amplification in dendritic cells via type I interferon-mediated activation of APOBEC3G.

Human immunodeficiency virus (HIV) is taken up by and replicates in immature dendritic cells (imDCs), which can then transfer virus to T cells, amplifying the infection. Strategies known to boost DC function were tested for their ability to overcome this exploitation when added after HIV exposure. Poly(I:C), but not single-stranded RNA (ssRNA) or a standard DC maturation cocktail, elicited type I interferon (IFN) and interleukin-12 (IL-12) p70 production and the appearance of unique small (15- to 20-kDa) fragments of APOBEC3G (A3G) and impeded HIV(Bal) replication in imDCs when added up to 60 h after virus exposure. Comparable effects were mediated by recombinant alpha/beta IFN (IFN-alpha/beta). Neutralizing the anti-IFN-alpha/beta receptor reversed poly(I:C)-induced inhibition of HIV replication and blocked the appearance of the small A3G proteins. The poly(I:C)-induced appearance of small A3G proteins was not accompanied by significant differences in A3G mRNA or A3G monomer expression. Small interfering RNA (siRNA) knockdown of A3G could not be used to reverse the poly(I:C)-induced protective effect, since siRNAs nonspecifically activated the DCs, inducing the appearance of the small A3G proteins and inhibiting HIV infection. Notably, the appearance of small A3G proteins coincided with the shift of high-molecular-mass inactive A3G complexes to the low-molecular-mass (LMM) active A3G complexes. The unique immune stimulation by poly(I:C) with its antiviral effects on imDCs marked by the expression of IFN-alpha/beta and active LMM A3G renders poly(I:C) a promising novel strategy to combat early HIV infection in vivo.

Novel (n)PKC kinases phosphorylate Nef for increased HIV transcription, replication and perinuclear targeting.

The N-terminus of the human immunodeficiency virus (HIV) pathogenicity factor Nef associates with a protein complex (NAKC for Nef-associated kinase complex) that contains at least two kinases: the tyrosine kinase Lck and a serine kinase activity which was found to phosphorylate Lck and the Nef N-terminus. Here we show that this serine kinase activity is mediated by members of the novel Protein Kinase C (nPKC) subfamily, PKCdelta and theta. Association with the Nef N-terminus was sufficient to activate PKC leading to phosphorylation of Nef in vitro on a conserved serine residue at position 6. Mutation of serine 6 or coexpression of a transdominant negative PKC mutant significantly reduced Nef-stimulated HIV transcription and replication in resting PBMC. When analyzing the molecular mechanisms, we found that mutating serine 6 moderately affected myristoylation of Nef and its association with Pak2 activity, whereas CD4 downmodulation was not inhibited. More interestingly, this mutation abolished the typical perinuclear localization of Nef in T cells. We conclude that the activation of nPKCs by Nef is required to increase viral replication/infectivity and direct the subcellular localization of Nef.

HIV-1 Nef enhances both membrane expression and virion incorporation of Env products. A model for the Nef-dependent increase of HIV-1 infectivity.

The expression of human immunodeficiency virus Nef increases the viral infectivity through mechanisms still not fully elucidated. Here we report that wild-type (wt) human immunodeficiency virus, type 1 (HIV-1), particles were neutralized by higher concentrations of either anti-Env glycoprotein (gp) 41 antibodies or recombinant soluble human CD4 compared with Deltanef HIV-1. This appeared to be the result of a Nef-induced increase of virion incorporation of both gp41 (transmembrane (TM)) and surface gp120 Env products likely originating from enhanced steady-state levels of cell membrane-associated Env products. This, in turn, seemed to be the consequence of a reduced retention of the Env precursor. Most interesting, we found that both the Nef-directed increase of Env membrane expression and the Nef-induced enhancement of HIV-1 infectivity relied on the presence of the intracytoplasmic domain of TM, supporting the hypothesis of a functional correlation between these effects. Mutagenesis studies allowed us to establish that the two leucine residues at the TM C terminus, which are part of a sorting motif involved in the control of Env membrane expression, and the 181-210-residue Nef C-terminal region were critically involved in the Nef/Env functional interaction. In conclusion, we propose that Nef increases the infectivity of HIV-1 at least in part by enhancing the amounts of Env products incorporated into virus particles.