21 research outputs found
A Stable Heroin Analogue That Can Serve as a Vaccine Hapten to Induce Antibodies That Block the Effects of Heroin and Its Metabolites in Rodents and That Cross-React Immunologically with Related Drugs of Abuse
An
improved synthesis of a haptenic heroin surrogate <b>1</b> (6-AmHap)
is reported. The intermediate needed for the preparation
of <b>1</b> was described in the route in the synthesis of <b>2</b> (DiAmHap). A scalable procedure was developed to install
the C-3 amido group. Using the Boc protectng group in <b>18</b> allowed preparation of <b>1</b> in an overall yield of 53%
from <b>4</b> and eliminated the necessity of preparing the
diamide <b>13</b>. Hapten <b>1</b> was conjugated to tetanus
toxoid and mixed with liposomes containing monophosphoryl lipid A
as an adjuvant. The <b>1</b> vaccine induced high anti-<b>1</b> IgG levels that reduced heroin-induced antinociception and
locomotive behavioral changes following repeated subcutaneous and
intravenous heroin challenges in mice and rats. Vaccinated mice had
reduced heroin-induced hyperlocomotion following a 50 mg/kg heroin
challenge. The <b>1</b> vaccine-induced antibodies bound to
heroin and other abused opioids, including hydrocodone, oxycodone,
hydromorphone, oxymorphone, and codeine
Efficacy, but Not Antibody Titer or Affinity, of a Heroin Hapten Conjugate Vaccine Correlates with Increasing Hapten Densities on Tetanus Toxoid, but Not on CRM<sub>197</sub> Carriers
Vaccines against drugs of abuse have
induced antibodies in animals
that blocked the biological effects of the drug by sequestering the
drug in the blood and preventing it from crossing the blood-brain
barrier. Drugs of abuse are too small to induce antibodies and, therefore,
require conjugation of drug hapten analogs to a carrier protein. The
efficacy of these conjugate vaccines depends on several factors including
hapten design, coupling strategy, hapten density, carrier protein
selection, and vaccine adjuvant. Previously, we have shown that <b>1</b> (MorHap), a heroin/morphine hapten, conjugated to tetanus
toxoid (TT) and mixed with liposomes containing monophosphoryl lipid
A [LÂ(MPLA)] as adjuvant, partially blocked the antinociceptive effects
of heroin in mice. Herein, we extended those findings, demonstrating
greatly improved vaccine induced antinociceptive effects up to 3%
mean maximal potential effect (%MPE). This was obtained by evaluating
the effects of vaccine efficacy of hapten <b>1</b> vaccine conjugates
with varying hapten densities using two different commonly used carrier
proteins, TT and cross-reactive material 197 (CRM<sub>197</sub>).
Immunization of mice with these conjugates mixed with LÂ(MPLA) induced
very high anti-<b>1</b> IgG peak levels of 400–1500 μg/mL
that bound to both heroin and its metabolites, 6-acetylmorphine and
morphine. Except for the lowest hapten density for each carrier, the
antibody titers and affinity were independent of hapten density. The
TT carrier based vaccines induced long-lived inhibition of heroin-induced
antinociception that correlated with increasing hapten density. The
best formulation contained TT with the highest hapten density of ≥30
haptens/TT molecule and induced %MPE of approximately 3% after heroin
challenge. In contrast, the best formulation using CRM<sub>197</sub> was with intermediate <b>1</b> densities (10–15 haptens/CRM<sub>197</sub> molecule), but the %MPE was approximately 13%. In addition,
the chemical synthesis of <b>1</b>, the optimization of the
conjugation method, and the methods for the accurate quantification
of hapten density are described
Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques
<div><p>HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24<sup>gag</sup> elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length <i>gag</i> DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55<sup>gag</sup> increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist.</p></div
Characteristics of p24CE-induced T cells.
<p>Plot overlays show the phenotypic and functional characterization of the CE-specific T cells induced by p24CE DNA vaccination (2 weeks after vaccination 2; upper panels) and after the heterologous p55<sup>gag</sup> boost (2 weeks after vaccination 3; lower panels) upon stimulation with CE-specific peptide pool. The analysis of macaque P307 is also shown in the absence of peptide stimulation (medium only) to illustrate the experimental background. The plots show overlays of total T cell population (grey contours) and the CE-specific IFN-γ<sup>+</sup> T cells (black dots) with (<b>A</b>) central memory (CM; CD28<sup>+</sup>CD95<sup>+</sup>) and effector memory (EM; CD28<sup>−</sup>CD95<sup>+</sup>) phenotype; (<b>B</b>) granzyme B production and (<b>C</b>) TNFα and CD107a expression. The numbers in the plots represent the frequencies of CE-specific IFN-γ<sup>+</sup> T cells. (<b>D</b>) Frequency of total CE-specific polyfunctional (4 functions; IFN-γ<sup>+</sup> TNF-α<sup>+</sup> CD107a<sup>+</sup> GzmB<sup>+</sup>) CE-specific T cells before and after the heterologous boost. P values are from non-parametric Mann-Whitney test.</p
Higher response rate and broader responses among the 7 CE in the p24CE DNA vaccinated macaques.
a<p>CE responses were evaluated 2 weeks after the priming vaccination shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086254#pone-0086254-t001" target="_blank">Table 1</a> using a peptide pool spanning the 7 individual CE (combination of 15-mer overlapping by 11AA and 10-mer overlapping by 9AA).</p
Single p55<sup>gag</sup> DNA boost of p24CE DNA vaccinated macaques elicits <i>de novo</i> cellular immune responses to regions outside of the CE.
a<p>analysis includes p2, p7, p1, p6 Gag peptides.</p
Boosting of p24CE DNA primed macaques with p55<sup>gag</sup> DNA increases CE-specific cellular responses.
<p>(<b>A</b>) Vaccination schedule of the animals primed with p24CE DNA (Vaccination 1 and 2) and boosted with p55<sup>gag</sup> DNA (vaccination 3). (<b>B</b>) Mapping of CE-specific T cell responses before (2 weeks after vaccination 2) and after (2 weeks after vaccination 3) the heterologous p55<sup>gag</sup> DNA boost of the 10 vaccinated macaques. The percentage of IFN-γ<sup>+</sup> CD4<sup>+</sup> (open bars) and CD8<sup>+</sup> (filled bars) T cells specific for each CE is shown. Note, although different scales were used for individual animals, the same scales are used before and after the DNA boost. Asterisks, indicates a sample that could not be analyzed. (<b>C</b>) Frequency of total CE-specific IFN-γ<sup>+</sup> T cells before and after the heterologous boost. P values were determined using non-parametric Mann-Whitney test.</p
p24CE DNA vaccine is immunogenic in macaques.
<p>(<b>A</b>) Alignment of the amino acid (AA) sequence of the 7 CE represented in the p24CE1 and p24CE2 proteins with HXB2 and COT-M p24<sup>gag</sup> proteins. The toggled AA in each CE is shown. The numbering of the AA in HXB2 p24<sup>gag</sup> protein is according to the HIVDB (<a href="http://www.hiv.lanl.gov/" target="_blank">www.hiv.lanl.gov/</a>). (<b>B</b>) The p24CE (p24CE1 and p24CE2) proteins are composed of 7 CE which were collinearly assembled in the order CE2-3-4-5-6-7-1 to avoid a strongly hydrophobic N-terminal CE1 were connected via short linker sequences designed for efficient proteolytic cleavage and contain the human GM-CSF signal peptide at the N-terminus as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086254#pone.0086254-Kulkarni1" target="_blank">[42]</a>. Plasmids SP-p24CE1 and SP-p24CE2 contain the GM-CSF signal peptide at the N-terminus of p24CE to promote secretion of the p24CE proteins. The CE1 and p24CE2 proteins differ by 1 AA per CE (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086254#pone-0086254-g001" target="_blank"><b>Figure 1A</b></a>), indicated by asterisks. (<b>C</b>) CE-specific cellular immune responses induced upon vaccination with p24CE. Macaques (N = 10) were vaccinated with p24CE DNA as outlined in the left panel and 2 weeks after the 2<sup>nd</sup> vaccination the cellular immune responses were analyzed. Note, macaques M437 and P317 received prior 3 vaccinations with DNAs expressing the poorly immunogenic unmodified p24CE (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086254#pone-0086254-t001" target="_blank">Table 1</a>). The frequency of CE-specific IFN-γ<sup>+</sup> T cells was measured using peptide pools composed of a mixture of 15-mer Group M peptides overlapping by 11 AA and 10-mer peptides overlapping by 9 AA covering both p24CE1 and p24CE2 proteins. The CE-specific CD4<sup>+</sup> (open bars) and CD8<sup>+</sup> (filled bars) T cells are shown.</p
Cellular immune responses in HIV-1 <i>gag</i> DNA vaccinated macaques.
a<p>analysis using peptide pool spanning p55<sup>gag</sup> (15-mer overlapping by 11 AA).</p>b<p>analysis using peptide pool spanning p24<sup>gag</sup> (15-mer overlapping by 11 AA).</p>c<p>analysis using peptide pool spanning all 7 CE (combination of 15-mer overlapping by 11 AA and 10-mer overlapping by 9 AA).</p
Changes of the CE response in macaques vaccinated with p55<sup>gag</sup> DNA after heterologous boost with p24CE DNA and upon co-immunization with p24CE and p55<sup>gag</sup> DNA.
<p>(<b>A</b>) Vaccination schedule of the animals primed with p55<sup>gag</sup> DNA and boosted with p24CE DNA. (<b>B</b>) Four of the p55<sup>gag</sup> DNA vaccinated animals, which developed cellular immune responses targeting CE (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086254#pone-0086254-t001" target="_blank"><b>Table 1</b></a>), received an additional vaccination with p24CE DNA. PBMC isolated 2 weeks after the 2<sup>nd</sup> p55<sup>gag</sup> DNA vaccination (vaccination 2) and after the p24CE DNA boost (vaccination 3) were stimulated with a CE-specific peptide pool (15- and 10-mer) covering individual 7 CE to map CE-specific T cell responses. The percentage of IFN-γ<sup>+</sup> CD4<sup>+</sup> (open bars) and CD8<sup>+</sup> (filled bars) T cells specific for each CE is shown. (<b>C</b>) Frequencies of the total CE-specific IFN-γ<sup>+</sup> T cells and (<b>D</b>) of the polyfunctional (4 functions; IFN-γ<sup>+</sup> TNF-α<sup>+</sup> CD107a<sup>+</sup> GzmB<sup>+</sup>) CE-specific T cells are shown before and after the heterologous boost. (<b>E</b>) Vaccination schedule of the animals co-immunized with p24CE and p55<sup>gag</sup> DNA. (<b>F</b>) PBMC isolated 2 weeks after the vaccinations 2 and 3 were stimulated with a CE-specific peptide pool covering the 7 individual CE. The percentage of IFN-γ<sup>+</sup> CD4<sup>+</sup> (open bars) and CD8<sup>+</sup> (filled bars) T cells specific for each CE is shown. The frequencies of the total (<b>G</b>) CE-specific IFN-γ<sup>+</sup> T cells and of the (<b>H</b>) polyfunctional (4 functions) CE-specific T cells are shown.</p