This paper models and experimentally measures the acoustic interference from two low intensity 3MHz continuous ultrasound transducers of the sam® wearable ultrasound device. Resulting data show that placement configuration, which dictates interference depth, and frequency phase variation are the main factors to acoustic pressure distribution. Mathematical analysis reveals that the acoustic pressure distribution from continuous ultrasound is modulated by near field variations at shallow tissue depths. This observation is useful in the application of sam® and in the further research of its therapeutic benefits.
We describe a drug delivery method that combines Time-Reversal Acoustics (TRA) with Convection-Enhanced Delivery (CED) to improve the delivery of therapeutics to the interstitium of the brain. The Ultrasound-assisted CED approach (UCED) circumvents the blood-brain barrier by infusing compounds through a cannula that is inserted into the brain while simultaneously delivering ultrasound to improve the penetration of pharmaceuticals. CED without ultrasound-assistance has been used to treat a variety of neural disorders, including glioblastoma multiforme, a malignancy that presents a very poor prognosis for patients. We describe a novel system that is used to infuse fluids into the brain parenchyma while simultaneously exposing the tissue to safe levels of 1-MHz, low intensity, ultrasound energy. The system includes a combined infusion needle-hydrophone, a 10-channel ultralow-output impedance amplifier, a broad-band ultrasound resonator, and MatLab®-based TRA control and user-interface. TRA allows easy coupling of ultrasound therapy through the skull without complex phase-correction and array design. The smart targeting UCED system has been tested in vivo and results show it provides 1.5-mm spatial resolution for UCED and improves tracer distribution in the brain over CED alone.
This paper explains the circuitry and signal processing to perform electrical impedance spectroscopy on piezoelectric materials and ultrasound transducers. Here, we measure and compare the impedance spectra of 2−5 MHz piezoelectrics, but the methodology applies for 700 kHz–20 MHz ultrasonic devices as well. Using a 12 ns wide 5 volt pulsing circuit as an impulse, we determine the electrical impedance curves experimentally using Ohm's law and fast Fourier transform (FFT), and compare results with mathematical models. The method allows for rapid impedance measurement for a range of frequencies using a narrow input pulse, digital oscilloscope and FFT techniques. The technique compares well to current methodologies such as network and impedance analyzers while providing additional versatility in the electrical impedance measurement. The technique is theoretically simple, easy to implement and completed with ordinary laboratory instrumentation for minimal cost.
impulse circuit; electrical impedance; piezoelectric; ultrasound measurement; spectroscopy; pulse
The structural flexibility found in human immunodeficiency virus (HIV) envelope glycoproteins creates a complex relationship between antigenicity and sensitivity to antiviral antibodies. The study of this issue in the context of viral particles is particularly problematic as conventional virus capture approaches can perturb antigenicity profiles. Here, we employed a unique analytical system based on fluorescence correlation spectroscopy (FCS), which measures antibody-virion binding with all reactants continuously in solution. Panels of nine anti-envelope monoclonal antibodies (MAbs) and five virus types were used to connect antibody binding profiles with neutralizing activities. Anti-gp120 MAbs against the 2G12 or b12 epitope, which marks functional envelope structures, neutralized viruses expressing CCR5-tropic envelopes and exhibited efficient virion binding in solution. MAbs against CD4-induced (CD4i) epitopes considered hidden on functional envelope structures poorly bound these viruses and were not neutralizing. Anti-gp41 MAb 2F5 was neutralizing despite limited virion binding. Similar antigenicity patterns occurred on CXCR4-tropic viruses, except that anti-CD4i MAbs 17b and 19e were neutralizing despite little or no virion binding. Notably, anti-gp120 MAb PG9 and anti-gp41 MAb F240 bound to both CCR5-tropic and CXCR4-tropic viruses without exerting neutralizing activity. Differences in the virus production system altered the binding efficiencies of some antibodies but did not enhance antigenicity of aberrant gp120 structures. Of all viruses tested, only JRFL pseudoviruses showed a direct relationship between MAb binding efficiency and neutralizing potency. Collectively, these data indicate that the antigenic profiles of free HIV particles generally favor the exposure of functional over aberrant gp120 structures. However, the efficiency of virion-antibody interactions in solution inconsistently predicts neutralizing activity in vitro.
HIV-1 infection of target cells is mediated via the binding of the viral envelope protein, gp120, to the cell surface receptor CD4. This interaction leads to conformational rearrangements in gp120 forming or revealing CD4 induced (CD4i) epitopes which are critical for the subsequent recognition of the co-receptor required for viral entry. The CD4-bound state of gp120 has been considered a potential immunogen for HIV-1 vaccine development. Here we report on an alternative means to induce gp120 into the CD4i conformation.
Combinatorial phage display peptide libraries were screened against HIV-1 gp120 and short (14aa) peptides were selected that bind the viral envelope and allosterically induce the CD4i conformation. The lead peptide was subsequently systematically optimized for higher affinity as well as more efficient inductive activity. The peptide:gp120 complex was scrutinized with a panel of neutralizing anti-gp120 monoclonal antibodies and CD4 itself, illustrating that peptide binding does not interfere with or obscure the CD4 binding site.
Two surfaces of gp120 are considered targets for the development of cross neutralizing antibodies against HIV-1; the CD4 binding site and CD4i epitopes. By implementing novel peptides that allosterically induce the CD4i epitopes we have generated a viral envelope that presents both of these surfaces simultaneously.
HIV-1; Vaccine; gp120; CD4i; Phage display; Peptide
The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs.
Recent studies have shown that natural infection by HIV-2 leads to the elicitation of high titers of broadly neutralizing antibodies (NAbs) against primary HIV-2 strains (T. I. de Silva, et al., J. Virol. 86:930–946, 2012; R. Kong, et al., J. Virol. 86:947–960, 2012; G. Ozkaya Sahin, et al., J. Virol. 86:961–971, 2012). Here, we describe the envelope (Env) binding and neutralization properties of 15 anti-HIV-2 human monoclonal antibodies (MAbs), 14 of which were newly generated from 9 chronically infected subjects. All 15 MAbs bound specifically to HIV-2 gp120 monomers and neutralized heterologous primary virus strains HIV-27312A and HIV-2ST. Ten of 15 MAbs neutralized a third heterologous primary virus strain, HIV-2UC1. The median 50% inhibitory concentrations (IC50s) for these MAbs were surprisingly low, ranging from 0.007 to 0.028 μg/ml. Competitive Env binding studies revealed three MAb competition groups: CG-I, CG-II, and CG-III. Using peptide scanning, site-directed mutagenesis, chimeric Env constructions, and single-cycle virus neutralization assays, we mapped the epitope of CG-I antibodies to a linear region in variable loop 3 (V3), the epitope of CG-II antibodies to a conformational region centered on the carboxy terminus of V4, and the epitope(s) of CG-III antibodies to conformational regions associated with CD4- and coreceptor-binding sites. HIV-2 Env is thus highly immunogenic in vivo and elicits antibodies having diverse epitope specificities, high potency, and wide breadth. In contrast to the HIV-1 Env trimer, which is generally well shielded from antibody binding and neutralization, HIV-2 is surprisingly vulnerable to broadly reactive NAbs. The availability of 15 human MAbs targeting diverse HIV-2 Env epitopes can facilitate comparative studies of HIV/SIV Env structure, function, antigenicity, and immunogenicity.
The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity.
In the RV144 trial, the estimated efficacy of a vaccine regimen against human immunodeficiency virus type 1 (HIV-1) was 31.2%. We performed a case–control analysis to identify antibody and cellular immune correlates of infection risk.
In pilot studies conducted with RV144 blood samples, 17 antibody or cellular assays met prespecified criteria, of which 6 were chosen for primary analysis to determine the roles of T-cell, IgG antibody, and IgA antibody responses in the modulation of infection risk. Assays were performed on samples from 41 vaccinees who became infected and 205 uninfected vaccinees, obtained 2 weeks after final immunization, to evaluate whether immune-response variables predicted HIV-1 infection through 42 months of follow-up.
Of six primary variables, two correlated significantly with infection risk: the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of HIV-1 envelope proteins (Env) correlated inversely with the rate of HIV-1 infection (estimated odds ratio, 0.57 per 1-SD increase; P = 0.02; q = 0.08), and the binding of plasma IgA antibodies to Env correlated directly with the rate of infection (estimated odds ratio, 1.54 per 1-SD increase; P = 0.03; q = 0.08). Neither low levels of V1V2 antibodies nor high levels of Env-specific IgA antibodies were associated with higher rates of infection than were found in the placebo group. Secondary analyses suggested that Env-specific IgA antibodies may mitigate the effects of potentially protective antibodies.
This immune-correlates study generated the hypotheses that V1V2 antibodies may have contributed to protection against HIV-1 infection, whereas high levels of Env-specific IgA antibodies may have mitigated the effects of protective antibodies. Vaccines that are designed to induce higher levels of V1V2 antibodies and lower levels of Env-specific IgA antibodies than are induced by the RV144 vaccine may have improved efficacy against HIV-1 infection.
The global transcriptional profile of peripheral blood mononuclear cells (PBMCs) stimulated with HIV candidate vaccine (virus-like particles, VLPs) has been evaluated in HIV-infected patients with low/high viral load compared to healthy volunteers. Baseline activation of chemokine production was observed in PBMC from HIV-infected patients and innate immune stimulation with HIV-VLPs was not blunted. The immune profile among HIV-infected patients was found to be qualitatively similar but quantitatively extremely variable. This diversity was independent of viral load and it might be dependent on individual immunogenetic traits or concurrent immunological status.
This ex vivo screening strategy represents an efficient tool for guiding modifications/optimizations of vaccination strategies and understanding failures in individuals enrolled in clinical trials.
Immunogenomics; Vaccine; HIV-1; Peripheral blood mononuclear cell
The β-chemokine RANTES (regulated on activation, normal T cell expressed and secreted) suppresses the infection of susceptible host cells by macrophage tropic strains of HIV-1. This effect is attributed to interactions of this chemokine with a 7-transmembrane domain receptor, CCR5, that is required for virus–cell fusion and entry. Here we identify domains of RANTES that contribute to its biological activities through structure–function studies using a new monoclonal antibody, mAb 4A12, isolated from mice immunized with recombinant human RANTES. This monoclonal antibody (mAb) blocked the antiviral activity of RANTES in infectivity assays with HIV-1Bal, and inhibited the mobilization of intracellular Ca2+ elicited by RANTES, yet recognized this chemokine bound to cell surfaces. Epitope mapping using limited proteolysis, reversed phase high-performance liquid chromatography, and mass spectrometry suggest that residues 55–66 of RANTES, which include the COOH-terminal α-helical region implicated as the glycosaminoglycan (GAG) binding domain, overlap the determinant recognized by mAb 4A12. This is supported by affinity chromatography studies, which showed that RANTES could be eluted specifically by heparin from a mAb 4A12 immunoaffinity matrix. Removal of cell surface GAGs by enzymatic digestion greatly reduced the ability of mAb 4A12 to detect RANTES passively bound on cell surfaces and abrogated the ability of RANTES to elicit an intracellular Ca2+ signal. Taken together, these studies demonstrate that the COOH-terminal α-helical region of RANTES plays a key role in GAG-binding, antiviral activity, and intracellular Ca2+ signaling and support a model in which GAGs play a key role in the biological activities of this chemokine.
β-chemokines; human immunodeficiency virus 1; monoclonal antibody; signaling; antiviral effect
Certain antibodies from HIV-infected humans bind conserved transition state (CD4 induced [CD4i]) domains on the HIV envelope glycoprotein, gp120, and demonstrate extreme dependence on the formation of a gp120-human CD4 receptor complex. The epitopes recognized by these antibodies remain undefined although recent crystallographic studies of the anti-CD4i monoclonal antibody (MAb) 21c suggest that contacts with CD4 as well as gp120 might occur. Here, we explore the possibility of hybrid epitopes that demand the collaboration of both gp120 and CD4 residues to enable antibody reactivity. Analyses with a panel of human anti-CD4i MAbs and gp120-CD4 antigens with specific mutations in predicted binding domains revealed one putative hybrid epitope, defined by the human anti-CD4i MAb 19e. In virological and immunological tests, MAb 19e did not bind native or constrained gp120 except in the presence of CD4. This contrasted with other anti-CD4i MAbs, including MAb 21c, which bound unliganded, full-length gp120 held in a constrained conformation. Conversely, MAb 19e exhibited no specific reactivity with free human CD4. Computational modeling of MAb 19e interactions with gp120-CD4 complexes suggested a distinct binding profile involving antibody heavy chain interactions with CD4 and light chain interactions with gp120. In accordance, targeted mutations in CD4 based on this model specifically reduced MAb 19e interactions with stable gp120-CD4 complexes that retained reactivity with other anti-CD4i MAbs. These data represent a rare instance of an antibody response that is specific to a pathogen-host cell protein interaction and underscore the diversity of immunogenic CD4i epitope structures that exist during natural infection.
To examine the relationship between HIV-1 antigenic load (plasma RNA copies/ml) and broad HIV-1 neutralizing antibody activity.
Plasma from 120 HIV-1 infected patients, including HIV-1 Natural Viral Suppressors (similar to Elite Controllers), was tested for neutralization against 15 Tier 1/Tier 2 HIV-1 pseudoviruses. Broad HIV-1 neutralizing antibody activity was confirmed with IgG and heterlogous clade testing (18 pseudoviruses from Clades A, C, and CRF02_AG). Statistical analysis was performed to determine factors associated with broad HIV-1 neutralizing antibody activity.
Ten individuals with broad HIV-1 neutralizing antibody activity were identified. These individuals had a median CD4 count of 589 cells/ul (range 202–927), 1,611 HIV-1 RNA copies/ml (range 110–8,964), and 13 years since HIV diagnosis (range 1–22). There was a significant correlation between the presence of broadly neutralizing antibodies in those with HIV-1 RNA between 100 and 10,000 copies/ml compared to those <100 or >10,000 copies/ml (p=.0003 and .0245, respectively). Individuals with HIV-1 RNA 100–10,000 copies/ml had a higher number of Tier 2 viruses neutralized compared to the <100or >10,000 copies/ml groups (p=< .0001 and p=.076, respectively). Male sex was associated with broad HIV-1 neutralizing antibody activity (p=.016).
These results indicate that low but persistent HIV antigen expression correlates with broad HIV-1 neutralizing antibody activity. At higher levels of plasma viremia, neutralization titers were diminished. Conversely, at lower levels, there appears to be insufficient antigen stimulation to maintain high neutralization titers. These findings may have important implications in furthering the understanding of the humoral response to HIV infection.
HIV; broadly neutralizing antibody; neutralizing activity; HIV RNA; natural viral suppressor; elite controller
Most DNA-encoded adjuvants enhance immune responses to DNA vaccines in small animals but are less effective in primates. Here, we characterize the adjuvant activity of the catalytic A1 domain of cholera toxin (CTA1) for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) antigens in mice and macaques delivered by GeneGun. The inclusion of CTA1 with SIVmac239 Gag dramatically enhanced anti-Gag antibody responses in mice. The adjuvant effects of CTA1 for the secreted antigen HIV gp120 were much less pronounced than those for Gag, as the responses to gp120 were high in the absence of an adjuvant. CTA1 was a stronger adjuvant for Gag than was granulocyte-macrophage colony-stimulating factor (GM-CSF), and it also displayed a wider dose range than GM-CSF in mice. In macaques, CTA1 modestly enhanced the antibody responses to SIV Gag but potently primed for a recombinant Gag protein boost. The results of this study show that CTA1 is a potent adjuvant for SIV Gag when delivered by GeneGun in mice and that CTA1 provides a potent GeneGun-mediated DNA prime for a heterologous protein boost in macaques.
Seventeen women who were persistently uninfected by human immunodeficiency virus type 1 (HIV-1), despite repeated sexual exposure, and 12 of their HIV-positive male partners were studied for antiviral correlates of nontransmission. Thirteen women had ≥1 immune response in the form of CD8 cell noncytotoxic HIV-1 suppressive activity, proliferative CD4 cell response to HIV antigens, CD8 cell production of macrophage inflammatory protein–1β, or ELISPOT assay for HIV-1–specific interferon-γ secretion. The male HIV-positive partners without AIDS had extremely high CD8 cell counts. All 8 male partners evaluated showed CD8 cell–related cytotoxic HIV suppressive activity. Reduced CD4 cell susceptibility to infection, neutralizing antibody, single-cell cytokine production, and local antibody in the women played no apparent protective role. These observations suggest that the primary protective factor is CD8 cell activity in both the HIV-positive donor and the HIV-negative partner. These findings have substantial implications for vaccine development.
We have developed a portable high power ultrasound system with a very low output impedance amplifier circuit (less than 0.3 Ω) that can transfer more than 90% of the energy from a battery supply to the ultrasound transducer. The system can deliver therapeutic acoustical energy waves at lower voltages than those in conventional ultrasound systems, because energy losses owing to a mismatched impedance are eliminated. The system can produce acoustic power outputs over the therapeutic range (greater then 50 W) from a PZT-4, 1.54 MHz, 0.75 in diameter piezoelectric ceramic. It is lightweight, portable, and powered by a rechargeable battery. The portable therapeutic ultrasound unit has the potential to replace “plug-in” medical systems and RF amplifiers used in research. The system is capable of field service on its internal battery, making it especially useful for military, ambulatory, and remote medical applications.
It is well established that paracrine secretion of anti-viral CCR5 ligands by CD8+ and CD4+ T cells can block the infection of activated CD4+ T cells by R5 and dual-tropic isolates of HIV-1. By contrast, because CD4+ T cells can be infected by HIV-1 and at least some subsets secrete anti-viral CCR5 ligands, it is possible that these ligands protect against HIV-1 via autocrine as well as paracrine pathways. Here we use a model primary CD4+ T cell response in vitro to show that individual CD4+ T cells that secrete anti-viral CCR5 ligands are ‘self-protected’ against infection with R5 but not X4 strains of HIV-1. This protection is selective for CD4+ T cells that secrete anti-viral CCR5 ligands in that activated CD4+ T cells in the same cultures remain infectable with R5 HIV-1. These data are most consistent with an autocrine pathway of protection in this system and indicate a previously unappreciated selective pressure on the emergence of viral variants and CD4+ T cell phenotypes during HIV-1 infection.
Baculovirus-expressed HIV-1 Pr55gag Virus-Like Particles (HIV-VLPs) induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with a production of Th1- and Th2-specific cytokines.
The analysis of genomic transcriptional profile of MDDCs, obtained from normal healthy donors and activated by HIV-VLPs, show the modulation of genes involved in the morphological and functional changes characterizing the MDDCs activation and maturation. Similar data are obtained using peripheral blood mononuclear cells (PBMCs), without further selection, showing the feasibility of a direct and “simplified” experimental procedure.
The results here described show that the maturation pattern induced by HIV-VLPs in ex vivo generated MDDCs, can be observed also in CD14-expressing freshly derived PBMCs, with the possible identification of genetic predictors of individual response to immunogens.
Infectious Agents and Cancer is a new open access, peer-reviewed, online journal, which encompasses all aspects of basic, clinical and translational research that provide an insight into the association between chronic infections and cancer.
Cholera toxin (CT) is a potent adjuvant that activates dendritic cells (DC) by increasing intracellular cyclic AMP (cAMP) levels. In vivo and in vitro, very small amounts of CT induce potent adjuvant effects and activate DC. We hypothesized that DC intoxicated by CT may release factors that enhance their own maturation and induce the maturation of toxin-free bystander DC. Through the use of mixed cultures and transwell cultures, we found that human monocyte-derived DC (MDDC) pulsed with CT or other cAMP-elevating agonists induce the maturation of bystander DC. Many DC agonists including CT increase the production of prostaglandin E2 (PGE2) and nitric oxide (NO). For this reason, we determined whether the actions of PGE2 or NO are involved in the maturation of MDDC induced by CT or dibutyryl-cAMP (d-cAMP). We found that blocking the production of PGE2 or blocking prostaglandin receptors inhibited MDDC maturation induced by CT and d-cAMP. Likewise, sequestering NO or blocking the downstream actions of NO resulted in the inhibition of MDDC maturation induced by CT and d-cAMP. These results indicate that endogenously produced factors including PGE2 and NO contribute to the maturation of DC induced by CT and that these factors participate in bystander DC maturation. The results of this study may help explain why bacterial toxins that elevate cAMP are such potent adjuvants.
We have recently developed a candidate HIV-1 vaccine model based on HIV-1 Pr55gag Virus-Like Particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from an Ugandan HIV-1 isolate of the clade A (HIV-VLPAs).
The HIV-VLPAs induce in Balb/c mice systemic and mucosal neutralizing Antibodies as well as cytotoxic T lymphocytes, by intra-peritoneal as well as intra-nasal administration. Moreover, we have recently shown that the baculovirus-expressed HIV-VLPs induce maturation and activation of monocyte-derived dendritic cells (MDDCs) which, in turn, produce Th1- and Th2-specific cytokines and stimulate in vitro a primary and secondary response in autologous CD4+ T cells.
In the present manuscript, the effects of the baculovirus-expressed HIV-VLPAs on the genomic transcriptional profile of MDDCs obtained from normal healthy donors have been evaluated. The HIV-VLPA stimulation, compared to both PBS and LPS treatment, modulate the expression of genes involved in the morphological and functional changes characterizing the MDDCs activation and maturation.
The results of gene profiling analysis here presented are highly informative on the global pattern of gene expression alteration underlying the activation of MDDCs by HIV-VLPAs at the early stages of the immune response and may be extremely helpful for the identification of exclusive activation markers.