Real-time nucleic acid sequence-based amplification using molecular beacons for detection of enterovirus RNA in clinical specimens.

Real-time nucleic acid sequence-based amplification (NASBA) using molecular beacon technology (NASBA-beacon) was compared to standard NASBA with postamplification hybridization using electrochemiluminescently labeled probes (NASBA-ECL) for detection of enteroviruses (EV) in 133 cerebrospinal fluid and 27 stool samples. NASBA-ECL and NASBA-beacon were similar in sensitivity, detecting 55 (100%) and 52 (94.5%) EV-positive samples, respectively. There were no false positives. Both NASBA assays were significantly more sensitive than culture. Real-time NASBA-beacon reagents and equipment rental were more expensive than those for NASBA-ECL; however, time to result was shortened by 1.5 h, hands-on time was reduced by 25 min, and the assay was much simpler for technologists to learn and perform.

Comparison of the NucliSens Basic kit (Nucleic Acid Sequence-Based Amplification) and the Argene Biosoft Enterovirus Consensus Reverse Transcription-PCR assays for rapid detection of enterovirus RNA in clinical specimens.

Samples were tested for enterovirus by nucleic acid sequence-based amplification (NASBA) (NucliSens Basic kit; BioMerieux), reverse transcription-PCR (RT-PCR) (Enterovirus Consensus RT-PCR kit; Argene Biosoft), and virus isolation. Eighty-two samples were tested, and 44 were positive, 34 by both NASBA and RT-PCR and 5 each by NASBA or RT-PCR only. Two nasopharyngeal samples positive only by RT-PCR were determined to be rhinovirus. Of 42 enterovirus-positive samples, NASBA detected 39 (92.9%) and RT-PCR detected 37 (88.1%). The NucliSens Basic kit and the Argene Biosoft RT-PCR had comparable sensitivities for detection of enterovirus RNA, and both molecular methods were more sensitive than culture, which detected only 60.5% of positive samples. NASBA could be completed in 6.5 h versus 9 h for the Argene Biosoft RT-PCR kit.

Rapid enterovirus RNA detection in clinical specimens by using nucleic acid sequence-based amplification.

Enterovirus (EV) detection by nucleic acid sequence-based amplification was compared with EV isolation in cell culture. The NucliSens Basic kit (bioMerieux) was utilized for RNA detection. For virus isolation, samples were inoculated into MRC-5, primary rhesus monkey kidney, A549, rhabdomyosarcoma, and/or Buffalo green monkey kidney cells.

Prospective analyses of HIV-1-specific proliferative responses, recall antigen proliferative responses, and clinical outcomes in an HIV-1-seropositive cohort.

BACKGROUND: In cross-sectional studies of chronically infected individuals, lymphoproliferative responses to human immunodeficiency virus (HIV) type 1 p24 Gag antigen have previously been associated with lower virus load. It was not known whether this association would be predictive of better clinical outcome in longitudinal studies. METHODS: In blood samples from 608 HIV-seropositive individuals enrolled in a trial of glycoprotein 160 vaccine therapy over the course of 3-5 years, lymphoproliferative responses to HIV-1 antigens, tetanus toxoid (TT), and mitogens were measured and correlated with clinical outcome and other parameters of progression. Baseline lymphoproliferative responses to antigens and mitogens were used to categorize the cohort into responders or nonresponders. RESULTS: Although response to recall antigens did not correlate with clinical indices of disease progression, positive baseline lymphoproliferative responses to p24 and TT were associated with lower plasma levels of HIV-1 RNA. Persistently positive lymphoproliferative responses to the antigens also inversely correlated with repeated measurements of virus load, although the significance was lost once the measurements were adjusted for virus load and CD4(+) cell count at baseline, by use of generalized estimating equation analysis. CONCLUSIONS: These observations suggest that the effect of the association between lymphoproliferative response and virus load is established early during HIV-1 infection and does not increase over time and suggest that antigen-specific lymphoproliferative responses reflect the dynamic state of HIV-1 infection and are inversely associated with virus load.

Immune reconstitution following autologous transfers of CD3/CD28 stimulated CD4(+) T cells to HIV-infected persons.

We have previously shown that adoptive transfer of in vitro CD3/CD28 activated autologous CD4(+) T cells results in increased CD4 counts and CD4/CD8 ratios in HIV+ subjects. In this report, analysis of variable beta (Vbeta) chain T cell receptor (TCR) repertoire showed that CD3/CD28 stimulation was able to increase polyclonality within skewed spectra types in vitro. In vivo, two of eight subjects showed increase in TCR diversity and importantly, in no subject did a highly skewed in vivo repertoire emerge. Measurement of proliferative response to alloantigen showed increases following infusions. Response to pharmacological stimulus and lectin via Interferon-gamma ELISpot assay showed increases in a subset of subjects following infusions. However, interferon-gamma response to HIV antigens and peptides declined concurrent with stable or diminishing latent infectious viral load in CD4(+) T cells. These data provide further evidence that adoptive transfer of activated autologous CD4(+) T cells can augment the immune system.

Safety and immunogenicity of an HIV subtype B and E prime-boost vaccine combination in HIV-negative Thai adults.

ALVAC-HIV (vCP1521) and AIDSVAX B/E were evaluated in a phase 1/2 trial of human immunodeficiency virus (HIV)-negative Thai adults. Of 133 volunteers enrolled, 122 completed the trial. There were no serious vaccine-related adverse events, nor were there intercurrent HIV infections. Lymphoproliferative responses to glycoprotein 120 E were induced in 63% of the volunteers, and HIV-specific CD8 cytotoxic T lymphocyte responses were induced in 24%. Antibody responses increased in frequency and magnitude in association with the dose level of AIDSVAX B/E. Binding and neutralizing antibodies to the MN strain were induced in 100% and 98%, respectively, of the volunteers receiving 600 microg of AIDSVAX B/E, and such antibodies to E strains were induced in 96% and 71%, respectively, of these volunteers. This vaccine combination was well tolerated and was immunogenic, meeting milestones for advancement to phase 3 evaluation.

Safety and immunogenicity of combinations of recombinant subtype E and B human immunodeficiency virus type 1 envelope glycoprotein 120 vaccines in healthy Thai adults.

Safety and immunogenicity of 2 recombinant human immunodeficiency virus (HIV) type 1 envelope glycoprotein (gp) 120 vaccines derived from SF2 (subtype B) and CM235 (CRF01_AE, Thai E) were evaluated in 370 Thai adults at low risk of HIV infection. Various doses of CM235 (25, 50, or 100 microg) and SF2 (0, 25, or 50 microg) gp120 were used. Eighty volunteers received placebo. There were no serious adverse events related to vaccination. Binding antibody developed in all vaccine recipients. There was no dose response to CM235 gp120, but a dose response to gp120 SF2 was present. Neutralizing antibodies to subtype E HIV-1 NPO3 and subtype B HIV-1 SF2 developed in 84% and 82% of vaccine recipients, respectively. Lymphoproliferative responses were detected in >95% of vaccine recipients. There was no evidence of antigenic interference in HIV-specific humoral or cellular responses. The gp120 Thai E and SF2 vaccines were safe and immunogenic in combination and could be advanced into phase 3 testing.