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1.
Nat Commun ; 11(1): 1483, 2020 Mar 20.
Article in English | MEDLINE | ID: mdl-32198363

ABSTRACT

Glasses are promising electrolytes for use in solid-state batteries. Nevertheless, due to their amorphous structure, the mechanisms that underlie their ionic conductivity remain poorly understood. Here, ab initio molecular dynamics is used to characterize migration processes in the prototype glass, 75Li2S-25P2S5. Lithium migration occurs via a mechanism that combines concerted motion of lithium ions with large, quasi-permanent reorientations of PS43- anions. This latter effect, known as the 'paddlewheel' mechanism, is typically observed in high-temperature crystalline polymorphs. In contrast to the behavior of crystalline materials, in the glass paddlewheel dynamics contribute to Lithium-ion mobility at room temperature. Paddlewheel contributions are confirmed by characterizing spatial, temporal, vibrational, and energetic correlations with Lithium motion. Furthermore, the dynamics in the glass differ from those in the stable crystalline analogue, γ-Li3PS4, where anion reorientations are negligible and ion mobility is reduced. These data imply that glasses containing complex anions, and in which covalent network formation is minimized, may exhibit paddlewheel dynamics at low temperature. Consequently, these systems may be fertile ground in the search for new solid electrolytes.

2.
Biotechniques ; 67(3): 126-137, 2019 09.
Article in English | MEDLINE | ID: mdl-31379198

ABSTRACT

Biological activity is a critical quality attribute for biopharmaceuticals, which is accurately measured using an appropriate relative potency bioassay. Developing a bioassay is a complex, rigorous undertaking that needs to address several challenges including modelling all of the mechanisms of action associated with the biotherapeutic. Bioassay development is also an exciting and fast evolving field, not only from a scientific, medical and technological point of view, but also in terms of statistical approaches and regulatory expectations. This has led to an industry-wide discussion on the most appropriate ways to develop, validate and control the bioassays throughout the drug lifecycle.


Subject(s)
Biological Products/pharmacology , Drug Evaluation, Preclinical/methods , Animals , Humans , Quality Control , Research Design
3.
ACS Appl Mater Interfaces ; 9(50): 43755-43766, 2017 Dec 20.
Article in English | MEDLINE | ID: mdl-29134805

ABSTRACT

One obstacle to realizing a practical, rechargeable magnesium-ion battery is the development of efficient Mg electrolytes. Electrolytes based on simple Mg(BH4)2 salts suffer from poor salt solubility and/or low conductivity, presumably due to strong ion pairing. Understanding the molecular-scale processes occurring in these electrolytes would aid in overcoming these performance limitations. Toward this goal, the present study examines the solvation, agglomeration, and transport properties of a family of Mg electrolytes based on the Mg(BH4)2 salt using classical molecular dynamics. These properties were examined across five different solvents (tetrahydrofuran and the glymes G1-G4) and at four salt concentrations ranging from the dilute limit up to 0.4 M. Significant and irreversible salt agglomeration was observed in all solvents at all nondilute Mg(BH4)2 concentrations. The degree of clustering observed in these divalent Mg systems is much larger than that reported for electrolytes containing monovalent cations, such as Li. The salt agglomeration rate and diffusivity of Mg2+ were both observed to correlate with solvent self-diffusivity: electrolytes using longer- (shorter-) chain solvents had the lowest (highest) Mg2+ diffusivity and agglomeration rates. Incorporation of Mg2+ into Mg2+-BH4- clusters significantly reduces the diffusivity of Mg2+ by restricting displacements to localized motion within largely immobile agglomerates. Consequently, diffusion is increasingly impeded with increasing Mg(BH4)2 concentration. These data are consistent with the solubility limitations observed experimentally for Mg(BH4)2-based electrolytes and highlight the need for strategies that minimize salt agglomeration in electrolytes containing divalent cations.

4.
PLoS One ; 11(7): e0160055, 2016.
Article in English | MEDLINE | ID: mdl-27467585

ABSTRACT

Chlamydia trachomatis is among the most prevalent of sexually transmitted diseases. While Chlamydia infection is a reportable event and screening has increased over time, enhanced surveillance has not resulted in a reduction in the rate of infections, and Chlamydia infections frequently recur. The development of a preventative vaccine for Chlamydia may be the only effective approach for reducing infection and the frequency of pathological outcomes. Current vaccine research efforts involve time consuming and/or invasive approaches for assessment of disease state, and MRI presents a clinically translatable method for assessing infection and related pathology both quickly and non-invasively. Longitudinal T2-weighted MRI was performed over 63 days on both control or Chlamydia muridarum challenged mice, either with or without elementary body (EB) immunization, and gross necropsy was performed on day 65. A scoring system was developed to assess the number of regions affected by Chlamydia pathology and was used to document pathology over time and at necropsy. The scoring system documented increasing incidence of pathology in the unimmunized and challenged mice (significantly greater compared to the control and EB immunized-challenged groups) by 21 days post-challenge. No differences between the unchallenged and EB immunized-challenged mice were observed. MRI scores at Day 63 were consistently higher than gross necropsy scores at Day 65, although two of the three groups of mice showed no significant differences between the two techniques. In this work we describe the application of MRI in mice for the potential evaluation of disease pathology and sequelae caused by C. muridarum infection and this technique's potential for evaluation of vaccines for Chlamydia.


Subject(s)
Chlamydia Infections/diagnostic imaging , Disease Models, Animal , Animals , Chlamydia Infections/microbiology , HeLa Cells , Humans , Magnetic Resonance Imaging , Mice
5.
Mediators Inflamm ; 2015: 264897, 2015.
Article in English | MEDLINE | ID: mdl-26663988

ABSTRACT

Chlamydia trachomatis is a bacterial sexually transmitted disease with over 1.3 million cases reported to the CDC in 2010. While Chlamydia infection is easily treated with antibiotics, up to 70% of infections are asymptomatic and go untreated. The current mouse model relies on invasive upper genital tract gross pathology readouts at ~60-80 days postinfection. High throughput optical imaging through the use of biomarkers has been successfully used to quickly evaluate several disease processes. Here we evaluate Neutrophil Elastase 680 (Elastase680) for its ability to measure Chlamydia muridarum associated inflammation in live mice using fluorescence molecular tomography (FMT) and In Vivo Imaging System (IVIS). Optical imaging was able to distinguish with statistical significance between vaccinated and nonvaccinated mice as well as mock-challenged and challenged mice 2 weeks after challenge which was 9 weeks sooner than typical gross pathological assessment. Immunohistochemistry confirmed the presence of neutrophils and correlated well with both in vivo and ex vivo imaging. In this report we demonstrate that Elastase680 can be used as a molecular imaging biomarker for inflammation associated with chlamydial infection in a mouse model and that these biomarkers can significantly decrease the time for pathology evaluation and thus increase the rate of therapeutics discovery.


Subject(s)
Chlamydia Infections/diagnosis , Chlamydia muridarum , Inflammation/diagnosis , Leukocyte Elastase/analysis , Animals , Biomarkers , Disease Models, Animal , Immunohistochemistry , Mice , Mice, Inbred C57BL , Vaccination
6.
Clin Vaccine Immunol ; 19(9): 1411-5, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22787198

ABSTRACT

Varicella-zoster virus (VZV)-specific cell-mediated immunity (CMI) responses were compared over time following an episode of herpes zoster (HZ) with those of age-, race-, and gender-matched healthy controls (HC) without HZ, using a validated gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay. The zoster brief-pain inventory (ZBPI) was used to assess zoster-associated pain. HZ patients (n = 140) had significantly higher IFN-γ ELISPOT responses to VZV antigen than did HC (n = 140). ELISPOT geometric mean count (GMC) responses (with 95% confidence intervals [CI]) for subjects who presented within 72 h were as follows: for HZ patients ≥ 60 years of age, at day 0 the GMC was 110 and at week 2 the GMC was 235; for HZ patients 21 to 59 years of age, at day 0 the GMC was 111 and at week 2 the GMC was 198; for HC ≥ 60 years of age, at day 0 the GMC was 19 and at week 2 the GMC was 18; and for HC 21 to 59 years of age, at day 0 the GMC was 59 and at week 2 the GMC was 56. The mean pain score (95% CI) across age groups at 1 week postrash (n = 106) was 6.0 (5.5, 6.5) and at 2 weeks postrash (n = 119) was 3.5 (2.9, 4.0). The percentage of HZ patients with substantial pain (score ≥ 3) at 6 weeks postrash increased with age from 8% for patients 21 to 49 years of age to 16% for patients 50 to 59 years of age to 22% for patients ≥ 60 years of age. The VZV-specific CMI response was substantially boosted by an episode of HZ, as measured by ELISPOT results. Older adults had lower VZV-specific cellular immunity than younger subjects at baseline, but the boosting effect of HZ was substantial for all age groups. HZ patients experienced considerable zoster-associated acute (1 to 2 weeks after rash) pain across age groups, while chronic pain increased with age.


Subject(s)
Enzyme-Linked Immunospot Assay/methods , Herpes Zoster/immunology , Herpes Zoster/pathology , Herpesvirus 3, Human/immunology , Herpesvirus 3, Human/pathogenicity , Pain/diagnosis , Adult , Age Factors , Aged , Aged, 80 and over , Female , Humans , Immunity, Cellular , Interferon-gamma/metabolism , Male , Middle Aged , Severity of Illness Index , Young Adult
7.
Vaccine ; 30(5): 904-10, 2012 Jan 20.
Article in English | MEDLINE | ID: mdl-22154769

ABSTRACT

BACKGROUND: Incidence and severity of herpes zoster (HZ) and postherpetic neuralgia increase with age, associated with age-related decrease in immunity to varicella-zoster virus (VZV). One dose of zoster vaccine (ZV) has demonstrated substantial protection against HZ; this study examined impact of a second dose of ZV. METHODS: Randomized, double-blind, multicenter study with 210 subjects ≥60 years old compared immunity and safety profiles after one and two doses of ZV, separated by 6 weeks, vs. placebo. Immunogenicity was evaluated using VZV interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISPOT) assay and VZV glycoprotein enzyme-linked immunosorbent antibody (gpELISA) assay. Adverse experiences (AEs) were recorded on a standardized Vaccination Report Card. RESULTS: No serious vaccine-related AEs occurred. VZV IFN-γ ELISPOT geometric mean count (GMC) of spot-forming cells per 10(6) peripheral blood mononuclear cells increased in the ZV group from 16.9 prevaccination to 49.5 and 32.8 at 2 and 6 weeks postdose 1, respectively. Two weeks, 6 weeks and 6 months postdose 2, GMC was 44.3, 42.9, and 36.5, respectively. GMC in the placebo group did not change during the study. The peak ELISPOT response occurred ∼2 weeks after each ZV dose. The gpELISA geometric mean titers (GMTs) in the ZV group were higher than in the placebo group at 6 weeks after each dose. Correlation between the IFN-γ ELISPOT and gpELISA assays was poor. CONCLUSIONS: ZV was generally well-tolerated and immunogenic in adults ≥60 years old. A second dose of ZV was generally safe, but did not boost VZV-specific immunity beyond levels achieved postdose 1.


Subject(s)
Herpes Zoster Vaccine/adverse effects , Herpes Zoster Vaccine/immunology , Vaccination/adverse effects , Vaccination/methods , Aged , Aged, 80 and over , Antibodies, Viral/blood , Double-Blind Method , Enzyme-Linked Immunosorbent Assay , Enzyme-Linked Immunospot Assay , Female , Herpes Zoster Vaccine/administration & dosage , Herpesvirus 3, Human/immunology , Humans , Interferon-gamma/metabolism , Male , Middle Aged , Placebos/administration & dosage
8.
J Infect Dis ; 200(7): 1068-77, 2009 Oct 01.
Article in English | MEDLINE | ID: mdl-19712037

ABSTRACT

BACKGROUND: The objectives of this study were to evaluate the association between varicella-zoster virus (VZV)-specific humoral and cell-mediated immunity (CMI) to herpes zoster (HZ) and protection against HZ morbidity and to compare immune responses to HZ and zoster vaccine. METHODS: In 981 elderly persons who developed HZ during a zoster vaccine efficacy trial (321 vaccinees and 660 placebo recipients) and 1362 without HZ (682 vaccinees and 680 placebo recipients), CMI was measured by VZV responder cell frequency and interferon-gamma enzyme-linked immunospot, and antibodies were measured by VZV enzyme-linked immunosorbent assay against affinity-purified VZV glycoproteins (gpELISA). RESULTS: Robust VZV CMI at HZ onset correlated with reduced HZ morbidity, whereas VZV gpELISA titers did not. Three weeks after HZ onset, gpELISA titers were highest in those with more severe HZ and were slightly increased in placebo recipients (compared with zoster vaccine recipients) and in older individuals. VZV CMI responses to HZ were similar in zoster vaccine and placebo recipients and were not affected by demographic characteristics or antiviral therapy, except for responder cell frequency at HZ onset, which decreased with age. When responses to zoster vaccine and HZ could be compared, VZV CMI values were similar, but antibody titers were lower. CONCLUSIONS: Higher VZV CMI at HZ onset was associated with reduced HZ severity and less postherpetic neuralgia. Higher antibody titers were associated with increased HZ severity and occurrence of postherpetic neuralgia. HZ and zoster vaccine generated comparable VZV CMI.


Subject(s)
Herpes Zoster/prevention & control , Herpesvirus 3, Human/immunology , Viral Vaccines/immunology , Aged , Antibodies, Viral/blood , Double-Blind Method , Female , Herpes Zoster/immunology , Humans , Immunity, Cellular , Male , Middle Aged , Neuralgia, Postherpetic/prevention & control , Time Factors
9.
Diagn Microbiol Infect Dis ; 63(2): 140-7, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19026505

ABSTRACT

Chlamydia trachomatis is a human pathogen that infects genital tracts in women. Disease control may be achieved through development of an efficacious vaccine. A mouse genital tract model serves as a tool for evaluation of vaccine candidates. Currently, assessment of infection in mice is performed by enumeration of inclusion-forming units (IFUs) through microscopic counting of fluorescently stained bacteria. We have developed a highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR) assay for enumeration of Chlamydia from mouse genital tracts to increase assay sensitivity, remove subjectivity, and improve sample throughput. The qPCR assay uses a 16S ribosomal gene sequence that is conserved across Chlamydia species and serovars, resulting in detection of multiple serovars of C. trachomatis, as well as Chlamydia muridarum and Chlamydia pneumoniae. The PCR assay provided results similar to IFU enumeration (94% agreement between the 2 assays) and is highly sensitive and specific with less inherent subjectivity than traditional enumeration methods.


Subject(s)
Bacterial Typing Techniques/methods , Chlamydia Infections/microbiology , Chlamydia/isolation & purification , Genitalia, Female/microbiology , Polymerase Chain Reaction/methods , Animals , Cell Line , Chlamydia/genetics , DNA Primers/genetics , DNA, Bacterial/genetics , Disease Models, Animal , Female , HeLa Cells , Humans , Mice , Mice, Inbred C57BL , RNA, Ribosomal, 16S/genetics , Reproducibility of Results , Sensitivity and Specificity , Vagina/microbiology
10.
J Immunol Methods ; 324(1-2): 84-91, 2007 Jul 31.
Article in English | MEDLINE | ID: mdl-17553519

ABSTRACT

Chlamydia trachomatis is an obligate intracellular pathogen that primarily infects epithelial cells. Traditional methods for quantification of inclusion forming units (IFUs) rely upon infection of epithelial cell monolayers in vitro. Following incubation for approximately 2 days, inclusion bodies that result from infection of cells are detected by immunofluorescent staining with an antibody conjugated to a fluorescent dye. These inclusion bodies are then manually counted by microscopic examination of multiple, randomly selected fields of view. This requires substantial operator time and is subject to investigator bias. We have developed a novel method in which we utilize an automated microplate ImmunoSpot reader to count C. trachomatis IFUs. Following infection of epithelial cells in a 96-well plate and subsequent incubation, IFUs are fixed and detected with an anti-C. trachomatis LPS monoclonal antibody. Immobilized antibody is detected with a biotinylated secondary antibody and visualized enzymatically with streptavidin-alkaline phosphatase and the colorimetric substrate nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phospate (NBT/BCIP). IFUs are then enumerated with the ImmunoSpot system. This method has been used to quantify IFUs from all cell lines traditionally used for chlamydial propagation, including L929, McCoy, HeLa and HaK cells. IFU numbers obtained are comparable to those determined by traditional microscopic counting. In addition, the method can be applied to rapid determination of serum-neutralizing titers for vaccine studies, and we have also applied this approach to quantify Chlamydia recovered from vaginal swabs collected from infected animals. This method provides for rapid enumeration of IFU counts while minimizing investigator bias and has potential applications for both research and diagnostic use.


Subject(s)
Chlamydia trachomatis/cytology , Chlamydia trachomatis/growth & development , Inclusion Bodies/microbiology , Animals , Cell Line , Cell Line, Tumor , Colony Count, Microbial/instrumentation , Colony Count, Microbial/methods , Cricetinae , HeLa Cells , Humans , Mice , Mice, Inbred BALB C
11.
Clin Vaccine Immunol ; 14(5): 527-37, 2007 May.
Article in English | MEDLINE | ID: mdl-17376862

ABSTRACT

The enzyme-linked immunospot (ELISPOT) assay is a powerful tool for measuring antigen-specific cellular immune responses. The ability to use frozen peripheral blood mononuclear cells (PBMC) facilitates testing samples in multicenter clinical trials; however, unreliable ELISPOT responses may result if samples are not handled properly. Exposure of frozen PBMC to suboptimal storage temperature (-20 degrees C) or repeated cycling between more optimal storage temperatures (less than -130 degrees C and -70 degrees C) reduced the quality of frozen PBMC, as assessed by cell viability and functional ELISPOT response measures. Cell viability as assessed by trypan blue dye exclusion was reduced, and the percentage of apoptotic cells, as determined by the Guava Nexin assay, was significantly increased after these events. The functional gamma interferon ELISPOT responses to phytohemagglutinin (PHA) mitogen, a CD4 T-cell-specific antigen (varicella-zoster virus), and a CD8 T-cell-specific antigen (pool containing known cytomegalovirus, Epstein-Barr virus, and influenza virus peptides) were all significantly reduced after suboptimal storage events. However, for a given suboptimal storage event, the magnitude of the reduction varied between individuals and even among aliquots within an individual bleed, indicating the need for sample-specific acceptance criteria (AC). The percent viable or percent apoptotic cells after thaw, as well as the functional ELISPOT response to PHA, were all effective when applied with limits as AC for separating samples damaged during storage from valid control samples. Although all three AC measures could be effectively applied, the apoptosis AC limit applied was best for separating samples that could respond to antigenic stimulation from samples that could not effectively respond.


Subject(s)
Cryopreservation , Enzyme-Linked Immunosorbent Assay/methods , Immunity, Cellular , Leukocytes, Mononuclear/immunology , Temperature , Cryopreservation/methods , Enzyme-Linked Immunosorbent Assay/standards , Humans , Interferon-gamma/analysis
12.
Vaccine ; 24(47-48): 6875-85, 2006 Nov 17.
Article in English | MEDLINE | ID: mdl-17050042

ABSTRACT

A new manufacturing process, known as process upgrade varicella vaccine (PUVV) was developed for a refrigerated formulation of varicella vaccine and for an investigational zoster vaccine. Safety and tolerability of a two-dose regimen of high-titered (approximately 50,000 PFU) PUVV were compared to a lower-titer formulation (approximately 5400 PFU) of VARIVAX; in 1366 healthy subjects > or =13 years old. Only one vaccine-related clinical serious adverse experience (pruritus; no hospitalization) was reported, in the VARIVAX group. Injection-site adverse experiences following any dose were higher in the PUVV group, 70.0%, than in the VARIVAX group, 56.2%, but generally were mild. Immunogenicity were similar in both groups in seronegative subjects. PUVV was generally well tolerated, and elicited an immune response similar to that induced by the marketed formulation of VARIVAX.


Subject(s)
Chickenpox Vaccine/adverse effects , Chickenpox Vaccine/immunology , Chickenpox/immunology , Chickenpox/prevention & control , Herpes Zoster/immunology , Herpes Zoster/prevention & control , Adolescent , Antibodies, Viral/analysis , Antibodies, Viral/biosynthesis , Antibody Formation/immunology , Chickenpox Vaccine/administration & dosage , Double-Blind Method , Female , Humans , Immunity, Cellular/immunology , Interferon-gamma/biosynthesis , Male
13.
J Virol ; 79(9): 5684-94, 2005 May.
Article in English | MEDLINE | ID: mdl-15827183

ABSTRACT

Measurements of serum-neutralizing antibody and anti-rotavirus immunoglobulin A (IgA) are the current standard for assessing immune responses following rotavirus vaccination. However, there is ongoing debate as to whether antibody titers correlate with protection against rotavirus gastroenteritis. Children recovering from rotavirus gastroenteritis have increased gamma interferon release from cultured peripheral blood mononuclear cells (PBMCs), suggesting that cell-mediated immunity (CMI) may play a role in viral clearance and protection from subsequent gastroenteritis. We have developed a gamma interferon enzyme-linked immunospot (ELISPOT) assay for evaluation of CMI responses to rotavirus using frozen PBMCs obtained from healthy adults. Responses to three different rotavirus antigen types were analyzed-a peptide pool based on the human VP6 sequence; reassortant human:bovine vaccine strains; and cell culture-adapted (CCA) human G1, G2, G3, G4, and bovine (WC3) G6 strains. The reassortant strains consist of a bovine WC3 genome background expressing the human rotavirus surface proteins VP7 (G1, G2, G3, or G4) or VP4 (P1). Responses to titrations of the peptide pool as well as CCA and reassortant strains were assessed. Gamma interferon ELISPOT responses were similar for CCA and reassortant strains, whether live or UV inactivated, and when tested either individually or pooled. For most subjects, responses to the VP6 peptide pool positively correlated with responses to CCA and reassortant strains. Cell depletion studies indicate the memory responses detected with these frozen adult PBMCs were primarily due to the CD4+ T-cell population. This gamma interferon ELISPOT assay provides a new tool to apply in clinical studies for the characterization of natural or vaccine-induced CMI to rotavirus.


Subject(s)
Immunoenzyme Techniques/methods , Immunologic Memory , Interferon-gamma , Rotavirus Infections/immunology , Rotavirus/immunology , T-Lymphocytes/immunology , Adult , Aged , Antigens, Viral/immunology , Cells, Cultured , Humans , Immunity, Cellular , Interferon-gamma/biosynthesis , Leukocytes, Mononuclear , Middle Aged , Sensitivity and Specificity
14.
J Infect Dis ; 188(9): 1336-44, 2003 Nov 01.
Article in English | MEDLINE | ID: mdl-14593591

ABSTRACT

The safety and immunogenecity of a booster dose of live attenuated varicella-zoster virus (VZV) vaccine was evaluated in 196 healthy subjects, >or=60 years old, who had already received a VZV vaccine >5 years before. This repeat booster dose was well tolerated. Cell-mediated immunity (CMI) to VZV was measured by an interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell (ELISPOT) assay and a limiting dilution responder cell frequency (RCF) assay. Prevaccination responses decreased as a function of increasing age but were detectable in all subjects by use of the IFN-gamma ELISPOT assay. In most subjects, VZV-specific CMI was increased at 6 weeks postvaccination. The magnitude of the vaccine-induced IFN-gamma ELISPOT response was inversely related to prevaccination values. Although there was a significant correlation between the IFN-gamma ELISPOT and RCF assays, the ELISPOT assay had greater sensitivity and a wider dynamic range. A live attenuated VZV vaccine is safe and immunogenic in an elderly population, and the vaccine-induced immunity may be monitored by the IFN-gamma ELISPOT assay.


Subject(s)
Herpesvirus 3, Human/immunology , Viral Vaccines/immunology , Age Factors , Aged , Aged, 80 and over , Antigens, Viral/immunology , Cell Division/immunology , Female , Herpes Zoster/immunology , Herpes Zoster/prevention & control , Humans , Immunity, Cellular/immunology , Immunization, Secondary , Immunoenzyme Techniques , Interferon-gamma/immunology , Interferon-gamma/metabolism , Male , Middle Aged , Sex Factors , Th1 Cells/immunology , Vaccines, Attenuated/immunology
15.
J Virol ; 77(11): 6305-13, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12743287

ABSTRACT

Cellular immune responses, particularly those associated with CD3(+) CD8(+) cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4(+) and CD8(+) T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.


Subject(s)
AIDS Vaccines/immunology , Genes, gag/immunology , Genetic Vectors/immunology , HIV Infections/prevention & control , Vaccines, DNA/immunology , AIDS Vaccines/administration & dosage , Adenoviruses, Human/genetics , Adenoviruses, Human/immunology , Adjuvants, Immunologic , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Genes, gag/genetics , HIV Antibodies/blood , HIV Infections/immunology , HIV-1/immunology , Humans , Immunization , Macaca mulatta , Plasmids , Recombination, Genetic , Vaccinia virus/genetics , Vaccinia virus/immunology , Virus Replication
16.
J Med Virol ; 70 Suppl 1: S38-41, 2003.
Article in English | MEDLINE | ID: mdl-12627485

ABSTRACT

An interferon-gamma ELISPOT assay has been developed for assessment of cellular immune responses to Varicella-Zoster Virus (VZV) in large, multi-center clinical vaccine trials. We show that the assay performed best when testing peripheral blood mononuclear cells (PBMCs) that had been isolated and then frozen on the same day as blood was drawn, and that freezing PBMCs from blood that was stored overnight before processing resulted in dramatically reduced responses. This assay was used to monitor cell-mediated immunity (CMI) in response to a booster immunization with an investigational live, attenuated VZV vaccine in an elderly population that had been vaccinated 8-10 years previously. The booster vaccine elicited a 1.6- to 1.7-fold rise in the VZV-specific cellular immune response as measured by the ELISPOT assay. The increase from pre to post booster vaccination response was more pronounced (approximately 2.2-fold rise) in a subset of subjects who had received two prior immunizations with a live, attenuated vaccine.


Subject(s)
Chickenpox Vaccine/administration & dosage , Enzyme-Linked Immunosorbent Assay/methods , Herpesvirus 3, Human/immunology , Immunity, Cellular , Interferon-gamma/analysis , Aged , Humans , Immunization, Secondary , In Vitro Techniques , Interferon-gamma/biosynthesis , T-Lymphocytes/immunology
17.
J Virol ; 76(19): 10038-43, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12208982

ABSTRACT

We examined the influence of dose and method of antigen delivery on the dynamics and durability of T-cell responses to candidate human immunodeficiency virus (HIV) vaccines. Codon-optimized sequences from the HIV gag gene were inserted into alternative DNA vaccine vectors to express the coding sequence with or without the tissue plasminogen activator leader sequence. We delivered the vaccines by intramuscular injection as plasmid DNA without adjuvant or as plasmid DNA formulated with a novel block copolymer adjuvant (CRL8623) and then monitored the ensuing T-cell responses by using a gamma interferon enzyme-linked immunospot assay. We demonstrated persistence of the cell-mediated immune (CMI) response in rhesus macaques for at least 18 months following a four-dose vaccination regimen. The plasmid vaccine, with or without CRL8623, was immunogenic in macaques; however, the form coadministered with adjuvant exhibited improved T-cell responses, with a bias toward more antigen-specific CD8(+) T cells. Finally, we examined the fine specificity of the T-cell response to the gag vaccines by testing the response of 23 vaccinated macaques to individual Gag 20-mer peptides. Collectively, the monkeys responded to 25 epitopes, and, on average, each monkey recognized a minimum of 2.7 epitopes. The results indicate that a broad and durable CMI response to HIV DNA vaccines can be induced in a relevant nonhuman primate model.


Subject(s)
AIDS Vaccines/immunology , Gene Products, gag/immunology , Interferon-gamma/biosynthesis , T-Lymphocytes/immunology , Vaccines, DNA/immunology , Animals , Immunization , Macaca mulatta
18.
J Virol ; 76(1): 185-94, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11739684

ABSTRACT

A synthetic gene consisting of the reverse transcriptase (RT) and integrase (IN) domains of human immunodeficiency virus type 1 (HIV-1) pol was constructed using codons most frequently used in humans. The humanized pol gave dramatically improved levels of Rev-independent, in vitro protein production in mammalian cells and elicited much stronger cellular immunity in rodents than did virus-derived gene. Specifically, BALB/c mice were immunized with plasmids and/or recombinant vaccinia virus constructs expressing the synthetic gene. High frequencies of Pol-specific T lymphocytes were detected in these animals by the gamma interferon enzyme-linked immunospot assay against pools of short overlapping peptides. Characterization of the stimulatory peptides from these pools indicates that the optimized gene constructs are able to effectively activate both CD4+ and CD8+ T cells. Immunization of rhesus macaques with DNA vaccines expressing the humanized pol coupled to a human tissue plasminogen activator leader sequence led to pronounced in vitro cytotoxic T-lymphocyte killing activities and enhanced levels of circulating Pol-specific T cells, comparable to those observed in HIV-1-infected human subjects. Thus, optimizing the immunogenic properties of HIV-1 Pol at the level of the gene sequence validates it as an antigen and provides an important step toward the construction of a potent pol-based HIV-1 vaccine component.


Subject(s)
AIDS Vaccines/administration & dosage , Genes, pol/immunology , HIV-1/immunology , Immunization , Vaccines, DNA/administration & dosage , AIDS Vaccines/immunology , Amino Acid Sequence , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Count , HIV-1/genetics , Humans , Macaca mulatta , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Plasmids , Spleen/cytology , Spleen/immunology , T-Lymphocytes, Cytotoxic/immunology , Time Factors , Vaccines, DNA/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology
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