Mutually exclusive T-cell receptor induction and differential susceptibility to human immunodeficiency virus type 1 mutational escape associated with a two-amino-acid difference between HLA class I subtypes

The relative contributions of HLA alleles and T-cell receptors (TCRs) to the prevention of mutational viral escape are unclear. Here, we examined human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) T-cell responses restricted by two closely related HLA class I alleles, B*5701 and B*5703, that differ by two amino acids but are both associated with a dominant response to the same HIV-1 Gag epitope KF11 (KAFSPEVIPMF). When this epitope is presented by HLA-B*5701, it induces a TCR repertoire that is highly conserved among individuals, cross-recognizes viral epitope variants, and is rarely associated with mutational escape. In contrast, KF11 presented by HLA-B*5703 induces an entirely different, more heterogeneous TCR beta-chain repertoire that fails to recognize specific KF11 escape variants which frequently arise in clade C-infected HLA-B*5703(+) individuals. These data show the influence of HLA allele subtypes on TCR selection and indicate that extensive TCR diversity is not a prerequisite to prevention of allowable viral mutations.     

Differential impact of simultaneous migration on coevolving hosts and parasites

Background  

The dynamics of antagonistic host-parasite coevolution are believed to be crucially dependent on the rate of migration between populations. We addressed how the rate of simultaneous migration of host and parasite affected resistance and infectivity evolution of coevolving meta-populations of the bacterium Pseudomonas fluorescens and a viral parasite (bacteriophage). The increase in genetic variation resulting from small amounts of migration is expected to increase rates of adaptation of both host and parasite. However, previous studies suggest phages should benefit more from migration than bacteria; because in the absence of migration, phages are more genetically limited and have a lower evolutionary potential compared to the bacteria.     

A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs

Background  

Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.     

Absolute requirement for an active immune response involving B cells and Th cells in immunity to Plasmodium yoelii passively acquired with antibodies to the 19-kDa carboxyl-terminal fragment of merozoite surface protein-1.

Vaccination of mice with the leading malaria vaccine candidate homologue, the 19-kDa carboxyl terminus of merozoite surface protein-1 (MSP119), results in sterile immunity to Plasmodium yoelii, with no parasites detected in blood. Although such immunity depends upon high titer Abs at challenge, high doses of immune sera transferred into naive mice reduce parasitemia (and protect from death) but do not result in a similar degree of protection (with most mice experiencing high peak parasitemias); this finding suggests that ongoing parasite-specific immune responses postchallenge are essential. We analyzed this postchallenge response by transferring Abs into manipulated but malaria-naive mice and observed that Abs cannot protect SCID, nude, CD4+ T cell-depleted, or B cell knockout mice, with all mice dying. Thus, in addition to the Abs that develop following MSP119 vaccination, a continuing active immune response postchallenge is required for protection. MSP119-specific Abs can adoptively transfer protection to strains of mice that are not protected following vaccination with MSP119, suggesting that the Ags targeted by the immune response postchallenge include Ags apart from MSP119. These data have important implications for the development of a human malaria vaccine.     

Sulfhydryl-alkylating reagents inactivate the NAD glycohydrolase activity of pertussis toxin

The combination of ATP, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), and DTT (dithiothreitol) is known to promote the expression of the NAD glycohydrolase activity of pertussis toxin, which resides in the toxin's S1 subunit. By monitoring changes in electrophoretic mobility, we have found that ATP and CHAPS act by promoting the reduction of the disulfide bond of the S1 subunit. In addition, ATP, CHAPS, and DTT allowed sulfhydryl-alkylating reagents to inactivate the NAD glycohydrolase activity. In the presence of iodo[14C]acetate, the combination of ATP, CHAPS, and DTT increased 14C incorporation into only the S1 subunit of the toxin, indicating that alkylation of this subunit was responsible for the loss of activity. If iodoacetate is used as the alkylating reagent, alkylation can be monitored by an acidic shift in the isoelectric point of the S1 peptide. Including NAD in alkylation reactions promoted the accumulation of a form of the S1 peptide with an isoelectric point intermediate between that of native S1 and that of S1 alkylated in the absence of NAD. This result suggests that NAD interacts with one of the two cysteines of the S1 subunit. In addition, we found the pH optimum for the NAD glycohydrolase activity of pertussis toxin is 8, which may reflect the participation of a cysteine in the catalytic mechanism of the toxin.     

Immunoaffinity chromatography using electroelution.

Immunoaffinity chromatography and electroelution of protein from solid-phase matrices are two powerful tools often used to purify proteins. In this study, we combined these two techniques and found that antigen was effectively recovered from immunoaffinity resins by electroelution. Yields ranged from 90.5% to 62.8%, with a mean of 74.0 +/- 7.4% (mean +/- standard deviation). A major portion of the eluate, 79.4 +/- 13.1%, was concentrated in a volume of 100 microliters and 94.0 +/- 2.0% was recovered in 200 microliters, even when 1 ml of resin was used. Electroelution had no major effect on the electrophoretic mobility of the antigen. Two distinct antigens, a relatively hydrophilic 230-kDa protein and a hydrophobic 28-kDa protein were successfully electroeluted. In addition two types of immunoaffinity resins, monoclonal antibody covalently linked to CNBr-activated Sepharose or immobilized protein A, were found to be compatible with this method.