Measles Virus Hemagglutinin epitopes immunogenic in natural infection and vaccination are targeted by broad or genotype-specific neutralizing monoclonal antibodies.

Measles virus (MV) remains a leading cause of vaccine-preventable deaths in children. Protection against MV is associated with neutralizing antibodies that preferentially recognize the viral hemagglutinin (MV-H), and to a lesser extent, the fusion protein (MV-F). Although MV is serologically monotypic, 24 genotypes have been identified. Here we report three neutralization epitopes conserved in the more prevalent circulating MV genotypes, two located in the MV-H receptor binding site (RBS) (antigenic site III) and a third in MV-H/MV-F interphase (antigenic site Ia) which are essential for MV multiplication. In contrast, two MV-H neutralization epitopes, showed a genotype-specific neutralization escape due to a single amino acid change, that we mapped in the "noose" antigenic site, or an enhanced neutralization epitope (antigenic site IIa). The monoclonal antibody (mAb) neutralization potency correlated with its binding affinity and was mainly driven by kinetic dissociation rate (koff). We developed an immunoassay for mAb binding to MV-H in its native hetero-oligomeric structure with MV-F on the surface of a MV productive steady-state persistently infected (p.i.) human cell lines, and a competitive-binding assay with serum from individuals with past infection by different MV genotypes. Binding assays revealed that a broad neutralization epitope, in RBS antigenic site, a genotype specific neutralization epitopes, in noose and IIa sites, were immunogenic in natural infection and vaccination and may elicit long-lasting humoral immunity that might contribute to explain MV immunogenic stability. These results support the design of improved measles vaccines, broad-spectrum prophylactic or therapeutic antibodies and MV-used in oncolytic therapies.

Prevention of nocturnal elevation of intraocular pressure by gene transfer of dominant-negative RhoA in rats.

IMPORTANCE: We developed a gene transfer tool for the control of nocturnal elevated intraocular pressure (IOP). OBJECTIVE: To demonstrate that inhibiting the trabecular meshwork RhoA pathway by delivering a mutated, dominant-negative RhoA gene (dnRhoA) carried inside a long-expressing recombinant virus would reduce nocturnal elevated IOP in a living animal. DESIGN AND SETTING: We generated an optimized recombinant viral molecule by inserting a mutated RhoA complementary DNA with a translation enhancer-promoter into a specially designed plasmid containing mutated viral terminal repeats. We then generated the virus particle, self-complementary adeno-associated virus serotype 2 carrying the mutated gene (scAAV2.dnRhoA) and assessed its function in vitro by infecting primary human trabecular meshwork cells and in vivo by injecting living rats intracamerally with therapeutic and control viruses. Three different models of 12-hour light and dark cycles were used. Viruses were injected when animals showed the circadian dark IOP elevation. The IOP measurements were conducted with a tonometer at 2 to 4 hours after onset of the nocturnal and diurnal cycles. Values at preinjection time were used as baselines. Animals were euthanized at 4 to 8 weeks after injection. EXPOSURES: Intraocular injection of rodent eyes with the recombinant viral vector scAAV2.dnRhoA. MAIN OUTCOMES AND MEASURES: Nocturnal elevation of IOP blocked for prolonged periods by transferred RhoA gene. RESULTS: By visual inspection, human trabecular meshwork cells infected with scAAV2.dnRhoA showed diminished stress fiber formation. Living rats exhibited a circadian IOP cycle that could be reset by adjusting light conditions to facilitate light and dark nocturnal IOP studies. A single-dose injection of scAAV2.dnRhoA into the rat eyes prevented elevation of IOP during the nocturnal cycle for at least 4 weeks (mean [SE], 9.2 [0.2] mm Hg light IOP and 9.6 [0.4] mm Hg dark IOP), while control eyes showed a significantly higher IOP over baseline (9.5 [0.4] mm Hg light IOP and 13.5 [0.3] mm Hg dark IOP). CONCLUSIONS AND RELEVANCE: To our knowledge, this is the first example of a gene transfer strategy that prevents nocturnal IOP elevation in living animals for prolonged periods. Inhibiting the RhoA pathway upstream of Rho kinase with a safe gene drug could provide a new enhanced treatment for long-term management of elevated nocturnal IOP.

A barrier-type insulator forms a boundary between active and inactive chromatin at the murine TCRß locus.

In CD4(-)CD8(-) double-negative thymocytes, the murine Tcrb locus is composed of alternating blocks of active and inactive chromatin containing Tcrb gene segments and trypsinogen genes, respectively. Although chromatin structure is appreciated to be critical for regulated recombination and expression of Tcrb gene segments, the molecular mechanisms that maintain the integrity of these differentially regulated Tcrb locus chromatin domains are not understood. We localized a boundary between active and inactive chromatin by mapping chromatin modifications across the interval extending from Prss2 (the most 3' trypsinogen gene) to D(ß)1. This boundary, located 6 kb upstream of D(ß)1, is characterized by a transition from repressive (histone H3 lysine 9 dimethylation [H3K9me2]) to active (histone H3 acetylation [H3ac]) chromatin and is marked by a peak of histone H3 lysine 4 dimethylation (H3K4me2) that colocalizes with a retroviral long terminal repeat (LTR). Histone H3 lysine 4 dimethylation is retained and histone H3 lysine 9 dimethylation fails to spread past the LTR even on alleles lacking the Tcrb enhancer (E(ß)) suggesting that these features may be determined by the local DNA sequence. Notably, we found that LTR-containing DNA functions as a barrier-type insulator that can protect a transgene from negative chromosomal position effects. We propose that, in vivo, the LTR blocks the spread of heterochromatin, and thereby helps to maintain the integrity of the E(ß)-regulated chromatin domain. We also identified low-abundance, E(ß)-dependent transcripts that initiate at the border of the LTR and an adjacent long interspersed element. We speculate that this transcription, which extends across D(ß), J(ß) and C(ß) gene segments, may play an additional role promoting initial opening of the E(ß)-regulated chromatin domain.

CD69 gene is differentially regulated in T and B cells by evolutionarily conserved promoter-distal elements.

CD69 is a type II C-type lectin involved in lymphocyte migration and cytokine secretion. CD69 expression represents one of the earliest available indicators of leukocyte activation and its rapid induction occurs through transcriptional activation. In this study we examined the molecular mechanism underlying mouse CD69 gene transcription in vivo in T and B cells. Analysis of the 45-kb region upstream of the CD69 gene revealed evolutionary conservation at the promoter and at four noncoding sequences (CNS) that were called CNS1, CNS2, CNS3, and CNS4. These regions were found to be hypersensitive sites in DNase I digestion experiments, and chromatin immunoprecipitation assays showed specific epigenetic modifications. CNS2 and CNS4 displayed constitutive and inducible enhancer activity in transient transfection assays in T cells. Using a transgenic approach to test CNS function, we found that the CD69 promoter conferred developmentally regulated expression during positive selection of thymocytes but could not support regulated expression in mature lymphocytes. Inclusion of CNS1 and CNS2 caused suppression of CD69 expression, whereas further addition of CNS3 and CNS4 supported developmental-stage and lineage-specific regulation in T cells but not in B cells. We concluded CNS1-4 are important cis-regulatory elements that interact both positively and negatively with the CD69 promoter and that differentially contribute to CD69 expression in T and B cells.

T-B+NK+ severe combined immunodeficiency caused by complete deficiency of the CD3zeta subunit of the T-cell antigen receptor complex.

CD3zeta is a subunit of the T-cell antigen receptor (TCR) complex required for its assembly and surface expression that also plays an important role in TCR-mediated signal transduction. We report here a patient with T(-)B(+)NK(+) severe combined immunodeficiency (SCID) who was homozygous for a single C insertion following nucleotide 411 in exon 7 of the CD3zeta gene. The few T cells present contained no detectable CD3zeta protein, expressed low levels of cell surface CD3epsilon, and were nonfunctional. CD4(+)CD8(-)CD3epsilon(low), CD4(-)CD8(+)CD3epsilon(low), and CD4(-)CD8(-)CD3epsilon(low) cells were detected in the periphery, and the patient also exhibited an unusual population of CD56(-)CD16(+) NK cells with diminished cytolytic activity. Additional studies demonstrated that retrovirally transduced patient mutant CD3zeta cDNA failed to rescue assembly of nascent complete TCR complexes or surface TCR expression in CD3zeta-deficient MA5.8 murine T-cell hybridoma cells. Nascent transduced mutant CD3zeta protein was also not detected in metabolically labeled MA5.8 cells, suggesting that it was unstable and rapidly degraded. Taken together, these findings provide the first demonstration that complete CD3zeta deficiency in humans can cause SCID by preventing normal TCR assembly and surface expression.

Some nuts are tougher to crack than others.

In this issue of Immunity, Oestreich et al. (2006) show that, during V(D)J recombination, RSSs may have distinct accessibility requirements. Some rely on an enhancer-intrinsic, general chromatin opening function, whereas others require enhancer-promoter interactions that direct local chromatin remodeling.

Regulation of the murine Ddelta2 promoter by upstream stimulatory factor 1, Runx1, and c-Myb.

Accessibility control of V(D)J recombination at Ag receptor loci depends on the coordinate activities of transcriptional enhancers and germline promoters. Recombination of murine Tcrd gene segments is known to be regulated, at least in part, by the Tcrd enhancer (Edelta) situated in the Jdelta2-Cdelta intron. However, there has been little characterization of promoters and other cis-acting elements that are activated by or collaborate with Edelta and that might function to regulate Tcrd gene recombination events. We now describe a strong promoter that is tightly associated with the murine Ddelta2 gene segment. EMSAs reveal that upstream stimulatory factor 1, Runx1, c-Myb, lymphoid enhancer binding factor 1, NF1, and E47 all interact with this promoter in vitro. Of these, upstream stimulatory factor 1, Runx1, and c-Myb appear necessary for full promoter activity in transiently transfected cells. Moreover, the same three factors were found to interact with the promoter in vivo by chromatin immunoprecipitation. We suggest that these factors play important roles as Edelta-dependent regulators of Ddelta2 accessibility in vivo. Consistent with the established roles of c-Myb and Runx factors in Edelta function, we detected low level, enhancer-independent activity of the Ddelta2 promoter in transient transfection experiments. We speculate that the Ddelta2 promoter may play a role as a weak, enhancer-independent regulator in vivo, and might contribute to residual Tcrd rearrangement in Edelta(-/-) mice.

Enforcing order within a complex locus: current perspectives on the control of V(D)J recombination at the murine T-cell receptor alpha/delta locus.

V(D)J recombination proceeds according to defined developmental programs at T-cell receptor (TCR) and immunoglobulin loci as a function of cell lineage and stage of differentiation. Although the molecular details are still lacking, such regulation is thought to occur at the level of accessibility of chromosomal recombination signal sequences to the recombinase. The unique and complex organization of the TCRalpha/delta locus poses intriguing regulatory challenges in this regard: embedded TCRalpha and TCRdelta gene segments rearrange at distinct stages of thymocyte development, there is a highly regulated progression of primary followed by secondary rearrangements involving Jalpha segments, and there are important developmental constraints on V gene segment usage. The locus therefore provides a fascinating laboratory in which to explore the basic mechanisms underlying developmental control. We provide here a current view of cis-acting mechanisms that enforce the TCRalpha/delta locus developmental program, and we emphasize the unresolved issues that command the attention of our and other laboratories.