Download A Therapeutic Dendritic Cell-Based Vaccine for HIV-1

BRIEF REPORT
A Therapeutic Dendritic Cell-Based
Vaccine for HIV-1 Infection
Felipe Garca,1 Núria Climent,1 Lambert Assoumou,6 Cristina Gil,1
Nuria González,3 José Alcam,3 Agathe León,1 Joan Romeu,4
Judith Dalmau,5 Javier Martnez-Picado,2,5 Jeff Lifson,8 Brigitte Autran,7
Dominique Costagliola,6 Bonaventura Clotet,4,5 Josep M Gatell,1
Montserrat Plana, and Teresa Gallart,1 for the DCV2/MANON07- AIDS
Vaccine Research Objective Study Groupa
At least 8 dendritic cell (DC) immunotherapy clinical trials for
human immunodeficiency virus type 1 (HIV-1) infection in
humans have been published [1–8]. Most of them found that
DC immunotherapy elicits some degree of immunological response. In the present double-blinded, controlled, clinical trial,
we vaccinated untreated patients with HIV-1 infection with
monocyte-derived dendritic cells (MD-DCs) pulsed with heatinactivated autologous virus.
1Infectious
A double-blinded, controlled study of vaccination of untreated
patients with chronic human immunodeficiency virus type 1
(HIV-1) infection with 3 doses of autologous monocyte-derived dendritic cells (MD-DCs) pulsed with heat inactivated
autologous HIV-1 was performed. Therapeutic vaccinations
were feasible, safe, and well tolerated. At week 24 after first
vaccination (primary end point), a modest significant decrease
in plasma viral load was observed in vaccine recipients, compared with control subjects (P 5 .03). In addition, the change
in plasma viral load after vaccination tended to be inversely
associated with the increase in HIV-specific T cell responses in
vaccinated patients but tended to be directly correlated with
HIV-specific T cell responses in control subjects.
Clinical trial.gov NCT00402142
Received 4 March 2010; accepted 9 November 2010.
Potential conflicts of interest: none reported.
Presented in part: 17th Conference on Retroviruses and Opportunistic Infections, San
Francisco, CA, 16–20 February 2010 (abstract 77).
a
Members of the study group are listed at the end of the text.
Reprints or correspondence: Dr Felipe Garca, Infectious Diseases Unit, Hospital Clnic,
Villarroel, 170, 08036 Barcelona, Spain ([email protected]).
The Journal of Infectious Diseases 2011;203:473–478
Ó The Author 2011. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
[email protected]
1537-6613/2011/2034-0001$15.00
DOI: 10.1093/infdis/jiq077
PATIENTS AND METHODS
Chronically HIV-1 infected patients with baseline CD41
T lymphocyte counts .450 cells/mm3, nadir CD41 T cell counts
.350 cells/mm3, and plasma viral loads .10,000 HIV-1 RNA
copies/mL were enrolled. Patients had not received combination
antiretroviral therapy (cART) for at least the 2 years before
enrollment. The coprimary end points were safety, change in
plasma viral load at week 24, and proportion of patients with
a decrease in plasma viral load of at least .5 log10 copies/mL at
week 24. Secondary end points were plasma viral load response
at weeks 16 and 48, proportion of patients who started therapy
(cART) based on a CD41 T cell count ,300 cells/mm3 in 2
determinations separated by at least 1 month, and changes in
CD41 T cell count and HIV-1–specific immune responses.
Untreated chronically HIV-infected patients were randomized
in a double-blind protocol to receive 3 doses of vaccine subcutaneously separated by 2 week intervals (weeks 0, 2, and 4) with
at least 8 3 106 MD-DCs pulsed with heat-inactivated autologous
virus (109 RNA copies/dose; DC-HIV group) or with nonpulsed
8 3 106 MD-DCs (DC-control group), according to the same
schedule. Patients were followed up for 48 weeks after the first
dose of vaccine (week 0). All the immunologic and virologic
determinations were performed in a blinded fashion. The study
was explained to all patients in detail, and all gave written informed consent. The study was approved by the institutional
ethical review board and by the Spanish Regulatory Authorities.
One week before administration of each dose of vaccine,
plasma monocyte samples were obtained and cultured for 7 days
to develop MD-DCs. Virus for pulsing was isolated from subjects
and expanded by culture in heterologous peripheral blood
monocyte cells at a median of 10 months (interquartile range
[IQR], 3–12 months) before the first vaccination. The virus isolates containing culture supernatants were collected and heatinactivated twice at 56°C for 30 min, concentrated by ultrafiltration (300KDa Vivaspin 20; Sartorious) to a final volume of 1 mL,
and stored frozen at 280°C until use for pulsing MD-DC. The
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Diseases Department, Hospital Clinic - HIV Development Program in
Catalonia, Institut d'Investigacions Biomédiques August Pi I Sunyer, University of
Barcelona, Spain; 2Institució Catalana de Recerca i Estudis Avanc
xats, Barcelona,
Spain; 3AIDS Immunopathogenesis Unit, Centro Nacional de Microbiología,
Instituto de Salud Carlos III, Madrid, Spain; 4Lluita contra la SIDA Foundation,
Hospital Germans Trias i Pujol, Badalona, Spain; 5IrsiCaixa Foundation - HIV
Development Program in Catalonia, Badalona, Spain; 6Clinical Epidemiology,
Therapeutic Strategies and Virology in HIV Infection, Institut National de la Santé
et de la Recherche Médicale U943 et Université Pierre et Marie Curie Université
Paris 06, Unité Mixte de Recherche en Santé 943, Hôpital Pitié-Salpêtriére, Paris,
France; 7Cellular Immunology Laboratory, Université Pierre et Marie Curie Univ.
Paris 06, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de
Recherche en Santé 945, Hôpital Pitié-Salpêtriére, Paris, France; and 8AIDS and
Cancer Virus Program, Science Applications International Corporation, Frederick,
and National Cancer Institute, Frederick, Maryland
group) according to protocol-specified criteria. Overall, the
vaccine was well tolerated, with asymptomatic enlargement of
local lymph nodes seen after vaccination in 3 patients (2 in the
DC-HIV-1 group 6–12 h after injections at weeks 0, 2, and 4, and
1 in the DC-control group after injection at week 0) and 1
episode of influenza-like symptoms (in 1 patient in the DC-HIV
group at week 0). The enlargement of lymph nodes regressed
after 48–72 h without intervention in each instance. Seven patients in the DC-HIV-1 group reported at least 1 adverse event
during the follow-up period, compared with 9 patients in the
DC-control group. Except for the lymph node enlargement and
the episode of influenza-like symptoms, the adverse events were
classified as unrelated to vaccination. The DC injections were
not associated with any clinical or serologic evidence of autoimmunity (data not shown).
Changes in Viral Load and CD41 T cell Count after DC
Vaccination
At weeks 16, 24, and 48, there was a median decrease in plasma
viral load of .23, .20, and .31 log10 RNA copies/mL in DC-HIV-1
RESULTS
Characteristics of Study Patients and Adverse Effects of
Vaccinations
Twenty-four patients were enrolled. The median age was 40
years (IQR, 34–46 years), and most of the patients (83%) were
men. In 67% of patients, the risk factor of HIV infection was
men who had sex with men. The median baseline of CD41
T cell count was 647 cells/mm3 (IQR, 532–776 cells/mm3), and
the median plasma viral load was 4.48 log10 copies/mL (IQR,
4.26–4.86 copies/ml. Baseline characteristics of the patients in
each arm were well balanced. Two patients in the DC-HIV-1
group were excluded from the analysis (one because of problems
with the preparation of the viral stock and the other because of
an unexpected decrease in CD4 T cell count; the latter patient
was removed before receiving any vaccine).
Three patients in the DC-HIV-1 group and 3 in the DCcontrol group started cART (at weeks 16, 16, and 36 in the DCHIV-1 group and at weeks 24, 36, and 36 in the DC-control
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Figure 1. Change in plasma viral load from baseline during the study
period. A, Median values. B, Individual values. Arrows represent
vaccinations. Numbers at bottom represent patients at risk. P values of
Mann-Whitney U test are shown at weeks 16, 24, and 48. P value of area
under the curve (AUC) is also shown.
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final products achieved an HIV-1 concentration .109 copies/mL,
a high infectivity reduction after heat-treatment (median, 5.5 log10
copies/mL), and were free from adventitious agents.
Serum neutralizing activity for CXCR4 and CCR5 viruses was
measured using a cell-based infectivity assay with recombinant
R5 (JR-FL) or X4 (pNL4.3) infectious HIV-1 clones harboring
the Renilla luciferase reporter gene, as described elsewhere [9].
Enzyme-linked immunospot (ELISPOT) assays were performed
to measure the number of interferon (IFN)–c-producing peripheral blood mononuclear cells (PBMCs) directed against
HIV-1 sequences. In brief, assays were performed with cryopreserved PBMC using 22 pools of peptides, consisting of 15mers overlapping by 11, grouped in pools of 10–12 peptides
each, covering the whole HIV-1 gag, nef, and env-gp41 sequences
(kindly provided by ORVACS), as described elsewhere [10].
Lymphoproliferative responses were performed as described
elsewhere [11].
The HIV RNA values were log10 transformed before analysis.
Measurements were censored after ART initiation in patients
who had to start ART according to preestablished criteria. The
continuous variables were compared between groups with use of
the nonparametric Mann-Whitney U test. The slopes of change
in CD4 cells count before vaccination with 3 measurements at
3-month intervals and after vaccination were compared using
a mixed linear regression model with random slopes for the time
before and after the first vaccination visit and with unstructured
matrix. Categorical variables were compared between groups with
use of the Fisher exact test. Changes in plasma HIV-1 RNA level
and the frequency of total HIV specific T cell responses over the 48
weeks of the trial were analyzed using an area-under-the-curve
(AUC) measurement. Spearman rank-order correlations were
calculated to assess the correlation between continuous variables.
Changes in HIV-1–Specific Responses after DC Vaccination
Neutralizing activity (NA) of serum was analyzed at weeks 0, 8,
and 24. The NA titers did not change significantly in DC-HIV-1
patients (median 50% inhibitory concentration [IC50], 1:41,
1:48, and 1:85 at weeks 0, 8, and 24, respectively) or in DCcontrol recipients (median IC50, 1:37, 1:17, and 1:34 at weeks
0, 8, and 24, respectively [P 5 .50, P 5 .78, P 5 .75, for the
comparison between groups at each time point, respectively]).
During vaccinations at weeks 2 and 4, there was a median
change in the frequency of total HIV-specific T cell responses
(defined as the sum of positive individual responses per patient) of -1249 and -202 SFC/106 PBMC in the DC-HIV-1 vs
35 and -17 SFC/106 PBMC in the DC-control group (P 5 .13
and P5 .96, respectively). After vaccinations, the median
change in the frequency of total HIV-specific T cell responses
in the DC-HIV-1 group, compared with that found in the DCcontrol group, was 632 vs -68 SFC/106 PBMCs (P 5 .82), 772
vs -969 SFC/106 PBMCs (P 5 .01), 332 vs -33 SFC/106 PBMCs
(P 5 .97), and 1370 vs 766 SFC/106 PBMCs (P 5 .68) at weeks
16, 20, 24, and 48, repsectively. Over a 48-week period, the
AUC analysis of the frequency of total HIV-specific T cell
responses showed a trend for a difference between the groups
(median, 720 [IQR , 281–1262] vs 1293 [IQR, 892–2120] in
the DC-control and DC-HIV, respectively; P 5 .10). The
increase in HIV-specific T cell responses observed after vaccination tended to be inversely associated with the decrease in
plasma viral load in vaccinated patients (q 5 -.54; P 5 .10),
although the change in HIV-specific T cell responses tended to
be directly correlated with changes in plasma viral load in
DC-control recipients (q 5 .53; P 5 .13) (Figure 2).
No positive HIV-1–specific CD4 lymphoproliferative responses to p24 antigen were observed in any patient.
DISCUSSION
The clinical trials published to date suggest that DC immunotherapy in HIV-1 infection is able to elicit HIV-specific immunological responses [1–8]. However, only 2 of these studies
reported virological responses to vaccination. A preliminary
noncontrolled, nonrandomized clinical trial reported by Lu et al
[2] was conducted in a population of cART-naive patients with
chronic HIV-1 infection and used autologous DCs pulsed with
whole aldrithiol-2 (AT-2)–inactivated autologous virus [2].
Those authors found that, after administration of 3 vaccine
doses, plasma viral load decreased by 90% for at least 1 year in 8
of 18 patients. This decrease in plasma viral load was associated
with strong, sustained HIV-1–specific cellular responses. Our
group performed an open, randomized (2:1), clinical trial in
patients who received ART with use of heat- inactivated autologous virus, finding partial and transient control of viral replication after interruption of ART [3]. In the present study, we
found that our autologous DC vaccine HIV-1 elicited only weak
HIV-1–specific T cell responses.
In the present study in patients who did not receive ART, we
found that this strategy was feasible, safe, and well tolerated. We
also found a modest virological response that was maintained
for at least 48 weeks in the vaccinated group, as suggested by the
AUC analysis. This change in plasma viral load in the vaccinated
patients, compared with the control group, was small (range,
.30–.60 log10 copies/mL) but was maintained from week 16 to
week 48. The small decrease in plasma viral load in vaccinated
patients tended to be inversely correlated with a modest increase
in HIV-1–specific T cell responses. In contrast, as other authors
have reported previously, in the control subjects, an observed
increase in HIV-1–specific T cells responses tended to correlate
directly with an increase in plasma viral load [11].
Although we repeated the same schedule of vaccinatins and
used a comparable dose of immunogen manufactured with
a similar procedure in HIV-1–infected patients with the same
clinical characteristics, the results of our clinical trial differ from
those reported by Lu et al [2]. The reasons for this difference are
not clear. Our trial was double blind and randomized, whereas
the trial by Lu et al [2] was open and not controlled. Another
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recipients, compared with an increase of .10, .21, and .34 log10
RNA copies/mL in DC-control recipients (P 5 .08, P 5 .03, and
P 5 .04, respectively) (Figure 1). The results of the analysis did
not change at week 24, when a sensitive analysis using last observation carried forward for the missing viral loads was performed (median change in plasma viral load ) 2.20 vs .14 log10
RNA copies/ml in DC-HIV-1 vs DC-control; P 5 .03). In the
patients in the DC-HIV-1 groups at weeks 16, 24, and 48, there
was a decrease in plasma viral load of >.5 log copies/mL in 3 of
10 (patients 13, 24, and 25), 1 of 8 (patient 24) and 2 of 6
patients (patients 13 and 24), respectively, in the DC-control
group, and 0 of 12, 0 of 11, and 0 of 9 patients showed a decrease
in plasma viral load of >.5 log copies/mL (P 5 .08, P 5 .42, and
P 5 .17, respectively). Over a 48-week period, the AUC analysis
showed a trend for a plasma viral load difference between the
groups (median, .14 [IQR, 2.21 to .45] vs 2.15 [IQR,2.47 to
.27] in DC-control and DC-HIV, respectively; P 5 .06).
Absolute values of CD4 and CD8 T cells did not change significantly during or after vaccinations. The median change in
CD41 T –cell count at weeks 16, 24, and 48 was 0, 26, and 276
cells/mm3 in the the DC-HIV-1 group and 14, 280, and 254
cells/mm3 in the DC-control group (P 5 .31, P 5 .13, and
P 5 1.00, respectively). To assess whether the decrease observed
in CD4 T cell count after vaccination was higher than would be
expected in the absence of vaccination, we compared the slope of
change in CD41 T cell count before and after the vaccinations.
We found no statistically significant differences between the
slopes (In DC-HIV-1 patients, 28.90 vs 24.92 [P 5 .16]; in
DC-control patients, 26.42 vs 24.92 [P 5 .88]).
Figrue 2. Correlation between the median change in the HIV-1-specific T cell responses with the median change in plasma viral load in patients in the
DC-HIV-1 group and DC-control recipients during the study period. Each dot represents a time point.
HOSPITAL CARLOS III DE MADRID (HCIII): José Miguel
Benito, Maria de la O López Vázquez de la Torre, Celia
Ballesteros Blanco.
d
HIVACAT-HOSPITAL CLÍNIC DE BARCELONA (HCB):
Teresa Gallart, Felipe Garcı́a, Nuria Climent, Cristina Gil,
Montserrat Plana, Agathe León, Llucia Alós Hernández, Miguel
Caballero Borrego, Alba Dı́az, Francisco Lomeña, José M Gatell.
d
HIVACAT-HOSPITAL GERMANS TRIAS I PUJOL.
BADALONA (HGTiP): Joan Romeu Fontanillas, Margarita Bofill
Soliguer, Christian Brander, Nuria Izquierdo, Judith Dalmau,
Javier Martinez-Picado, Bonaventura Bonaventura Clotet.
d
HOSPITAL GREGORIO MARAÑÓN DE MADRID
(HGUGM): Louis Chonco, Nickola Wever, Marjorie Pion,
Marı́a Jesús Serramı́a, Miguel Relloso, Paula Ortega, Javier de la
Mata, Rafael Gómez, Marı́a Ángeles Muñoz-Fernández.
d
HOSPITAL UNIVERSITARIO REINA SOFI´A DE
CÓRDOBA (HRS): José Peña Martı́nez, Rafael González
Fernández, Mario Frias Casas, Barbara Manzanares Martı́n y
Laura Castro.
d
INSTITUTO DE SALUD CARLOS III. MADRID
(ISCIII): Nuria González Fernandez, José Alcamı́, Ma Teresa
Perez Olmeda, Javier Garcia Perez, Luis Miguel Bedoya del
Olmo.
d
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Funding
This study was partially supported by grants: FIS PS09/01297, FIPSE
36750/08, SAF2008-04395, SAF 05/05566, FIPSE 36536/05, FIS 040503,
FIS 070291, contract FIS 03/0072, Mutua Madrileña del Automóvil,
STREP (UE) Life sciences, genomics and biotechnology for health
LSH2005_2.3.0.10, PROFIT (FIT 090100-2005-9), MARATÓ TV3, RIS**,
ORVACS***.
Dr Felipe Garcı́a was recipient of a Research Grant from IDIBAPS****,
Barcelona, Spain.
Dr. M. Plana was supported by contract FIS 03/0072 from the Fundació
Privada Clı́nic per a la Recerca Biomédica in collaboration with the
Spanish Health Department
Dr. Lifson is supported with federal funds from the National Cancer
Institute, National Institutes of Health, under Contract No.
HHSN261200800001E.
*FIPSE is a non-profit Foundation including: Spanish Ministry of
Health, Abbott Laboratories, Boehringer Ingelheim, Bristol Myers Squibb,
GlaxoSmithKline, Merck Sharp and Dohme and Roche).
**Red Temática Cooperativa de Grupos de Investigación en Sida del
Fondo de Investigación Sanitaria (FIS).
***ORVACS: Objectif recherche vaccin sida.
****IDIBAPS: Institut d’Investigacions Biomádiques August Pi I Sunyer
The authors do not have a commercial or other association that might
pose a conflict of interest.
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alternative explanation could be the inactivation method of the
virus used for pulsing MD-DCs. AT-2 inactivation preserves the
conformational and functional integrity of virion surface proteins, including envelope glycoproteins, which may improve
virus uptake by MD-DCs, allowing inactivated virus to enter
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APPENDIX
METHODS
Preparation of Inactivated Autologous Viral Stocks for
Pulsing Autologous MD-DC
For each subject, one lot of autologous inactivated HIV-1
stock was prepared according to clinical grade good
manufacturing practice (cGMP). Briefly, for each infected
patient a primary virus isolate was obtained and propagated by
means of co-culture of 25 3 106 CD4 enriched PBMCs from
both the HIV-1 infected subject along with 25 3 106 CD3pre-activated CD4-enriched PBMCs obtained from HIVseronegative unrelated healthy volunteer, during 21 days in
X-Vivo 20 media, 10% of AB human serum and IL-2. The virus
containing culture supernatants were collected and heatinactivated twice at 56°C for 30 minutes and concentrated by
ultrafiltration (300KDa Vivaspin 20, Sartorious) to a final
volume of 1 mL, which was divided into five .2 mL aliquots
(contained a median (IQR) Viral Load of 9.1 (7.9–9.77) Log10
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dilutions in PBMC cell cultures for 11 days, by using
a modified procedure previously described elsewhere (1).
The productive infection was determined by ELISA HIV
Ag p24. A high infectivity reduction after heat-treatment
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cell culture in PBMCs, MRC-5 and VERO cell lines,
microbiology cultures, gram and mycoplasma tests. A 515
bp fragment of the protease coding region of the virus present
in the patient plasma at the moment of autologous HIV-I
isolation sampling and the virus contained in the immunogen
were analyzed, No significant differences between were found
between both viruses.
Ex vivo Generation of MD-DCs, Their Pulsing With
Inactivated HIV-1 and Maturation Under cGMP Conditions,
and Immunizations
PBMC were isolated, within 1 hr of the blood extraction,
from a 150-mL sample of EDTA-treated venous blood by
means of standard Ficoll gradient centrifugation. After
washing (4x) in cGMP PBS (Lonza, Walkersville, Maryland,
USA), PBMC were resuspended (3 millions/mL) in MD-DC
culture medium, which consisted of serum-free X-VIVO15
culture medium (Lonza) supplemented with 1% of heatinactivated autologous serum (HI-AS), 50 microg/mL of
phamaceutical Gentamicin (B/Braun Medical), 2.5 microg/
mL of phamaceutical Fungizone (Bristol-MyersSquibb),
1 lM of phamaceutical zidovudine (Retrovir from
GlaxoSmithKline), and then, settled down in sterile
apyrogenic culture flasks (Corning, NY, USA). The reason
for adding zidovudine was to avoid endogenous HIV-1
replication. After 2 h. of incubation (at 37°C in a humidified
atmosphere of 5%-C02 in air), non-adherent cells were
discarded, and adherent cells (>95% CD141) washed (3x) in
pre-warmed (at 37 °C) cGMP PBS to eliminate residual
contaminating lymphocytes, and then differentiated into
immature MDDC in a 5-day culture with a total volume of
18 mL of the above MD-DC medium containing GMP
recombinant human (rh) GM-CSF (1000 IU/mL) and IL-4
(1000 IU/mL) (both cytokines from CellGenix, Freiburg,
Germany); these cytokines at the indicated concentration
were also added at day 2. On day 5 of culture, MD-DCs were
collected, washed (3x) with cGMP PBS and then resuspended
(>8 3 106) in a final volume of 3 mL of MD-DC medium
(with GM-CSF and IL-4 (1000 IU/mL each) and pulsed with
heat-inactivated autologous HIV-1 (.2 mL with .108 copies
of HIV-1 RNA) for 2-3 hr in a cell incubator. Thereafter, 22
mL of MD-DC medium (also containing GM-CSF and IL-4,
1000 IU/mL each) were added and the cell culture
prolonged for 45-46 hr, in the presence of the maturation
cocktail, which consisted of GMP rh IL-1 b (300 IU/mL), IL-6
(1000 IU/mL) and TNF-a (1,000 IU/mL) (all cytokines
from CellGenix). Afterward, MD-DCs were washed once
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and resuspended in .5 ml of pharmaceutical saline solution
containing 1% of pharmaceutical human Albumin (from
Grifols, Spain) and immediately injected subcutaneously in
the upper-inner part of both arms (.25 ml each injection).
Before, injection a small aliquot of MD-DCs, was used to
assess the yield, viability and immunophenotype of MD-DC
generated and to perform, Mycoplasma detection and
Gram staining Before pulsing, microbiological culture was
performed with uniform negative results. The amount (>8 3
106 MD-DCs), purity (>90%), viability (>85%), and the
maturation status (CD80 and CD83 markers >40%),
as assessed by flow cytometry, were consistent throughout
all the immunizations and fulfilled the predetermined
specifications approved by the Spanish Agency of
Medications for the current trial with the autologous
DC-based vaccine loaded with autologous inactivated
HIV-1, considered as an Investigational New Drug.
Immunophenotyping of MD-DCs by flow cytometry was
done as already reported (2)
Neutralizing Activity Assay
Serum neutralizing activity for CXCR4 and CCR5 viruses was
measured by using a cell-based infectivity assay with
recombinant R5 (JR-FL) or X4 (pNL4.3) infectious HIV-1
clones harboring the Renilla luciferase reporter gene. Viral
stocks were generated by transfection in 293 T-cells and titrated
in the U87CD4 cell line. In neutralization assays a viral dose
equivalent to 30.000 RLU/well was incubated for 1 h with serial
4-fold dilutions of sera before infection of either the
U87CD4CXCR4 or the U87CD4CCR5 cells. Virus infectivity
was determined 48 hours post-inoculation by measuring
luciferase activity in cell lysates. HIV neutralization was
calculated for each dilution. Using the formula: % inhibition 5
[(luciferase 1 Serum)/(luciferase – Serum)] 3 100 Sigmoid
curves were constructed and IC50 neutralizaiton titers (1/dilution
which confers 50% inhibition) were calculated by non-linear
regression using GraphPad v3.0 software.
ELISPOT Assay
ELISPOT assays were performed to measure the numbers of
IFN-c producing PBMC directed against HIV-1 sequences.
Briefly, assays were performed with cryopreserved PBMC
using 22 pools of peptides, consisting of 15-mers overlapping
by 11, grouped in pools of 10–12 peptides each, covering the
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Lymphoproliferative Responses
PBMC were washed twice and resuspended at 2 3 106/mL in
serum-free medium X-VIVO 10 (BioWhittaker, Maryland).
Cultures were plated in triplicate at 2 3 105/well in 7 day assays,
in 96 round-bottomed microplates (TPP, Europe). Cells were
cultured in the absence or presence of 10lg/ml Pokeweed
mitogen (PWM) (Sigma) as a polyclonal stimulus or 5 lg/mL
of recombinant HIV-1 proteins gp160, and p24 (Protein
Sciences, Meriden, CT). Incorporation of tritium-labeled
thymidine during the last 18h of 7-day culture was evaluated
using a betaplate scintillation counter (LKB Wallac, Finland).
Results were expressed as mean counts per minute (cpm). The
stimulation index (SI) was calculated for each sample as: cpm
for cells with stimulus/cpm for cells without stimulus. Positive
antigen-specific responses were defined as more than 3000 cpm
and SI greater than 3.
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Plasma Viral Load
Plasma viral load was analyzed by Versant HIV-1 RNA 3.0
Assay (b-DNA, Siemens Healthcare Diagnostics).
whole HIV-1 gag, nef and env-gp41 sequences (kindly provided
by ORVACS). Negative control responses were obtained with
unstimulated cells. Positive controls included cells stimulated
with phytohemaglutinin A (PHA, Sigma) and a CEF pool
containing 32 HLA-class I restricted peptides from CMV, EBV
and Flu virus (NIH AIDS Research and Reference Research
Program). All time-points for an individual patient were tested
in a single assay. The ELISPOT assay was done in 96-well PVDF
microtiter plates coated overnight with a mAb specific for
human IFN-c (mAb B-B1, Diaclone, BioNova, Spain). PBMC
resuspended in RPMI plus 10% FCS were plated in the presence
of different peptide pools (2 microg/ml, final concentration)
and incubated overnight at 37°C, 5% CO2. Plates were
developed using biotinylated anti-human IFN-c, streptavidin
conjugated to alkaline phosphatase (Amersham Biosciences,
UK) and chromogenic substrate BCIP/NBT (Sigma). Spot
forming cells (SFC) were counted using an AID ELISPOT
reader (Autoimmun Diagnostica GmHb, Germany). Results
were expressed as the number of spots-forming cells (SFC)
per million of PBMC after substracting the background.
A threshold for positive responses on the IFNgamma
ELISPOT was set for 50 SFC/106 cells after substracting SFC
present in negative control which should be at least ,50 SFC/
106 PBMC for being considered. Positive responses by
ELISPOT were only considered when results were .50 FC/
106 PBMC and at least 2 times the background. According to
Kinloch-de Loes et al (3), with this methodology the 85% at
least of the responding cells are CD8 T cells, but as all studies
were conducted with unfractionated PBMC, responses are
described as HIV-specific T cells.