Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL COMBINATION FOR THE TREATMENT OF AIDS
FIELD OF INVENTION
The present invention relates to a novel method for treating acquired immune
deficiency
syndrome (AIDS) in a subject in need thereof. In particular, said method
comprises the
administration of a combination, a kit-of-parts, a composition or a
pharmaceutical
composition comprising a type III interferon blocking agent. an interferon-
alpha (IFN-a)
blocking agent, an antiretroviral (ART) agent and, optionally, an interferon-
beta (IFN-13)
blocking agent and/or a latency-reversing agent (LRA).
BACKGROUND OF INVENTION
Antiretroviral therapy (ART) is thus far, a very efficient therapy in the
treatment of
patients infected with human immunodeficiency viruses (HIV). Indeed, patients
treated
with ART have a plasma viremia below detectable levels and thus can have
normal life
expectancy. However, these treatments are very onerous and can lead to
uncertain long-
term cytotoxicity for HIV patients (Chun et al., "Durable Control of HIV
Infection in the
Absence of Antiretro viral Therapy: Opportunities and Obstacles" JAMA,
2019 Jul 2;322(1):27-28).
In addition, the most considerable issue with ART is that when patients
stopped their
treatment and are thus in an analytic treatment interruption (ATI), a rapid
rebound of
plasma viremia is observed. This phenomenon suggests that not all HIV-infected
cells are
eliminated by ART and that some infected cells remain in the organism. The
scientific
community is thus wondering how and where HIV is able to persist in patient
during
ART. Two different types of cells were identified: latent and productive
infected cells. In
patients treated with ART, latent cells were identified in many T-cells types
(including
stem cell memory, central memory, transitional memory, effector memory and
naïve CD4
T cells), suggesting that these cells can contribute to viral rebound after
ART arrest, but
this mechanism still remains poorly understood (Pitman et al., "Barriers and
strategies to
achieve a cure for HIV" Lancet HIV, 2018 Jun;5(6):e317-e328).
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Under ART treatment, lymph nodes and gastrointestinal tract have much higher
concentration of HIV DNA and RNA per CD4 T cells than other tissues (e.g.
blood).
Indeed, in lymph nodes, CD8 cytotoxic T cells have less access to B-cell
follicles and
thus these cells are protected from the treatment, while in the
gastrointestinal tract, many
Th17 cells expressing CCR5 are present, cells targeted preferentially by the
virus, which
can explain the high level of HIV DNA and RNA. Macrophages from different
tissues
can also be infected and thus persist for example in the brain and this
population was
demonstrated as being the viral source of the viral rebound after ART arrest
in a unique
model of humanized myeloid-only mice (Honeycutt et al., "HIV persistence in
tissue
macrophages of humanized myeloid-only mice during antiretroviral therapy'
Nat Med, 2017; 23: 638-43).
Furthermore, the study of Rabezanahary et al., found that in the spleen
mesenteric and
peripheral lymph nodes of SIVmac251-infected rhesus macaques, effector memory
and
follicular helper T CD4 have the highest frequency of viral DNA and RNA.
Interestingly,
two weeks after ART arrest, a viral rebound was observed due to a rapid
seeding of SIV
from visceral lymphoid tissues producing viral RNA, highlighting the
importance of these
anatomical sites, reservoirs of HIV, in the viral rebound (Rabezanahary et
al., "Despite
early antiretroviral therapy effector memory and follicular helper CD4 T cells
are major
reservoirs in visceral lymphoid tissues of SIV-infected macaques" Mucosal
Immunology,
2020 13:149-160). Many other clinical strategies were investigated including
targeting
the viral replication cycle (BM transplantation of patients with hematopoietic
stem cells
from donors homozygous for CCD5A32 or WT CCR5, gene therapy by editing CCR5,
latency reversal agents). HIV-specific immune enhancement strategies (T-cell
vaccines,
bNAbs and CART cells) or immune modulation strategies (immune-checkpoint
blockers,
vedolizumab, IL15 superagonist or sirolimus) (Pitman et al., "Barriers and
strategies to
achieve a cure for HIV" Lancet HIV, 2018 Jun;5(6):e317-e328).
The HIV-1 latent reservoir is a major hurdle to achieve a cure for HIV-1. A
new strategy
consists in targeting the latent cells which escape the ART and can thus
create a viral
rebound after ART arrest. One hypothesis was that the HIV integrated mostly
into
heterochromatin-repressed transcriptional regions into these latent cells, but
it was
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surprisingly found that HIV can be latent while integrated in euchromatin-
active
transcriptional regions, suggesting that the epigenetic silencing occurs via
cis- or
trans- acting elements. So far, the therapies were focused on epigenetic
mechanisms of
latency and mostly histone deacetylase inhibitors (HDAC) inhibitors, but no
clear
reservoir reduction has been demonstrated. T-cell activating agents were also
a new wave
of therapy using for example protein kinase C (PKC) agonists but despite
promising
results in vitro, no reduction in the latent reservoir was reported in vivo.
One strategy
could also be a "shock and kill" strategy wherein the "shock" phase will be to
target the
immune response towards the reservoir and to reverse latency and then to kill
the cells,
but this strategy requires to know which antigen will be presented by the re-
activated cell.
Many others strategies are currently under investigation (Sengupta et al.,
"Targeting the
Latent Reservoir for HIV-1" Immunity, 2018 15;48(5):872-895 and Ait-Ammar et
al.,
"Current Status of Latency Reversing Agents Facing the Heterogeneity of HIV-1
Cellular
and Tissue Reservoirs" Frontiers in Microbiology, 2020 24;10:3060).
Although several latency-reversing agents (LRAs) have been identified and
tested, none
of them has been able to efficiently eradicate the HIV-1 latent reservoir.
Alternative therapeutic approaches aimed at reversing HIV latency in presence
of
antiretroviral therapy have been tested. One of them was aimed at controlling
the effects
of type I interferons, cytokines known to have a crucial role in the
immunopathogenesis
of AIDS during chronic HIV-1 infection. IFNAR blockade in the presence of cART
was
found to reduce the size of HIV-1 reservoirs in lymphoid tissues in humanized
mice
persistently infected with HIV-1, but was insufficient to efficiently
eradicate these
reservoirs.
The "shock and kill" strategy is based on inducing viral transcription of
latent HIV-1
provirus followed by the selective killing of reactivated cells.
Inefficient eradication of the HIV-1 latent reservoir is due to the
persistence of resting
cells containing latent HIV-1 provirus, since only reactivated cells are
killed by
antiretroviral (ART) agents in the "kill" phase of the "shock and kill"
strategy.
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In summary, latent HIV reservoir represents the main obstacle to achieving
sustained
virologic remission in ART-treated HIV-infected individuals following ART
treatment
interruption. And the reservoir cell compartment still needs to be better
understood in
order to improve the cure strategy against HIV infection.
Thus, there is still an important and urgent need for a novel treatment of HIV
infection or
acquired immune deficiency syndrome (AIDS) which allows sustained or complete
virologic remission in ART-treated HIV patients.
This could be achieved by finding new strategies that induce reactivation of
cells
constituting the HIV-1 latent reservoirs and viral transcription of latent HIV-
1 provirus
in these cells, or for therapeutic approaches that reverse mechanisms that
inhibit such
HIV-1 reactivation in the latent reservoirs.
Interferons are a group of cytokines of different types. Type I interferons
are systemic,
while type III interferons are mainly mucosal (i.e. produced at the mucosal
epithelial).
Based on their discovery of new biological properties of type I and type III
interferons,
the inventors propose to improve existing therapeutic strategies for treating
AIDS caused
by HIV.
In particular, they have shown that the production of type T IFN-ot and type
III IFN-X,
locally is induced by viral replication occurring in the peripheral and
mucosal reservoir
cells (which contain latent integrated HIV proviruses), and is involved in the
incomplete
virus clearance.
Indeed, the inventors have surprising shown the presence of type III
interferons in the
serum of patients after antiretroviral (cART) treatment, while the serum level
of type I
IFN had totally decreased after treatment. This antiviral cytokine is locally
produced by
mucosal cells, which are still infected and replicating the virus.
By their antiviral effects, these interferons locally reduce the ongoing viral
replication
occurring in these reservoir cells. This limits the local propagation of the
virus to nearby
cells, but at the time maintains the reservoir of infected cells.
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The inventors have thus surprisingly shown that reactivation of the HIV-1
latent
reservoirs is inhibited by type III interferons which are naturally produced
by HIV 1
infected patients, especially when under antiretroviral treatment.
Moreover, the inventors have surprisingly shown that this inhibition of HIV
reactivation
5 in the latently infected cells may be reversed by type III interferon
blocking agents.
The inventors thus provide a new therapeutic approach based on the use of a
type In
interferon blocking agent to reverse the natural or therapeutically-induced
mechanisms
that inhibit HIV-1 reactivation in the latent reservoirs.
This new therapeutic approach, combined with the "kill" phase of the "shock
and kill"
strategy performed by antiretroviral therapy (ART), will allow complete viral
clearance
from the latent reservoirs.
The inventors thus propose to block the type III interferon-k, the type I
interferon-a, and
optionally type I TFN interferon-I3 antiviral action in resistant cells
containing proviral
HIV-1 DNA in the HIV reservoirs, by repeated administration of specific agents
blocking
the production or of the activity of these cytokines, to reverse proviral
latency, while
administering ART agents to the patient until total viral clearance.
Indeed, following the blocking of the peripheral and mucosal antiviral
interferons, which
hinder expression of viral replication in reservoir cells, the HIV-1 proviral
DNA present
in these reservoir cells may fully replicate viruses. In turn, cART treatment,
which
controls viral replication, may reduce and progressively eliminate these
peripheral and
mucosal cellular reservoirs.
The applicant thus provides a novel method for treating AIDS in a subject in
need thereof,
comprising administering to the subject a combination comprising:
i) a type III interferon blocking agent,
ii) an interferon-alpha (IFN-a) blocking agent,
iii) optionally, an interferon-beta (IFN-11) blocking agent,
iv) an antiretroviral (ART) agent, and
v) optionally, a latency-reversing agent (LRA).
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SUMMARY
The invention relates to a combination comprising:
i) a type III interferon blocking agent,
ii) an interferon-alpha (IFN-a) blocking agent,
iii) optionally, an interferon-beta (IFN-f3) blocking agent,
iv) optionally, an antiretroviral (ART) agent, and
v) optionally, a latency-reversing agent (LRA).
In some embodiments, the type III interferon blocking agent is:
- an agent neutralizing circulating IFN-k selected from the group comprising
an active
anti-IFN-X vaccine, such as an IFN-k-kinoid, IFN-X, DNA-based or IFN-X RNA-
based
vaccine, and a passive anti-IFN-X vaccine, such as an anti-lFN-X antibody or
an anti-IFN-
X hyper-immune serum, or
- an agent blocking type III interferon signaling selected from the group
consisting of an
active anti-IFNLR1 vaccine, such as an IFNLR1 DNA-based or IFNLR1 RNA-based
vaccine, and a passive anti-IFNLR1 vaccine, such as an antibody that binds to
the
1FNLR1 receptor.
In some embodiments, the type III interferon blocking agent is an anti-IFN-X
antibody,
or an agent blocking type III interferon signaling selected from the group
consisting of an
active anti-IFNLR1 vaccine, such as an IFNLR1 DNA-based or IFNLR1 RNA-based
vaccine, and a passive anti-IFNLR1 vaccine, such as an antibody that binds to
the
1FNLR1 receptor.
In some embodiments, the IFN-a blocking agent is selected from the group
consisting of
an agent neutralizing circulating IFN-ox, an agent blocking IFN-a signaling,
an agent
depleting IFN-cc producing cells, and an agent blocking IFN-a production;
wherein the agent neutralizing circulating IFN-a is selected from the group
comprising an active anti-IFN-a vaccine, such as an IFN-a-kinoid, IFN-ct DNA-
based or
IFN-ot RNA-based vaccine, and a passive anti-IFN-a vaccine, such as an anti-
IFN-a
antibody or anti-IFN-a hyper-immune serum;
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wherein the blocking agent of IFN-a signaling is selected from the group
consisting of an active anti-IFNAR1 or anti-IFNAR2 vaccine, such as an IFNAR1
or
1FNAR2 DNA-based or an IFNAR1 or 1FNAR2 RNA-based vaccine, a passive anti-
IFNAR1 or anti-IFNAR2 vaccine, such as an anti-type T interferon R1 or R2
antibody,
and an IFN-a endogenous regulator, such as SOSC1 or an aryl hydrocarbon
receptor;
wherein the agent depleting IFN-a producing cells is an agent depleting
plasmacytoid dendritic cells (pDCs); and
wherein the agent blocking IFN-a production is an agent blocking the
production of IFN-
a by pDCs.
In some embodiments, the IFN-13 blocking agent is an agent neutralizing
circulating IFN-
13 selected from the group comprising an active anti-IFN-0 vaccine, such as an
1FN-f3-
kinoid, IFN-13 DNA-based or IFNI. RNA-based vaccine, and a passive anti-IFN-13
vaccine, such as an anti-IFN-13 antibody or an anti-IFN-13 hyper-immune serum.
In some embodiments, the antiretroviral (ART) agent is selected from the group
consisting of Nucleoside reverse transcriptase inhibitors (NRTIs), Non-
nucleoside
reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (PIs),
Integrase inhibitors
(MST's), Fusion inhibitors (FIs), Chemokine receptor antagonists (CCR5
antagonists)
and Entry inhibitors (CD4-directed post-attachment inhibitors).
In some embodiments, the latency-reversing agent (LRA) is selected from the
group
consisting of PKC agonists, MAPK agonists, CCR5 antagonists, Tat vaccines,
SMAC
mimetics, inducers of P-TEFb release, activators of Akt pathway, benzotriazole
derivatives, epigenetic modifiers and immunomodulatory LRAs.
Another aspect of the invention relates to a combination as described herein
for use in the
treatment of acquired immune deficiency syndrome (AIDS) in a subject in need
thereof.
In some embodiments, the presence of cells containing replication-competent
proviral
HIV DNA is assessed in a blood sample from the subject.
In some embodiments, the type III interferon blocking agent, the IFN-a
blocking agent,
optionally the interferon-beta (IFN-I3) blocking agent, the antiretroviral
(ART) agent, and
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optionally the latency-reversing agent are for simultaneous, separate or
sequential
administration.
In some embodiments, the type III interferon blocking agent, the IFN-a
blocking agent,
and optionally the interferon-beta (IFN-p) blocking agent are to be
administered every
week, every 2 weeks or every 3 weeks.
In some embodiments, the antiretroviral (ART) agent, and optionally the
latency-reversing agent, are to be administered daily.
In some embodiments, the type III interferon blocking agent, the IFN-a
blocking agent,
and optionally the interferon-beta (IFN-13) blocking agent are to be
administered
parenterally or intravenously.
In some embodiments, one or more doses of the type III interferon blocking
agent, the
IFN-ot blocking agent, and optionally the interferon-beta (IFN-P) blocking
agent are to be
administered to the subject before receiving said combination.
In some embodiments, one or more doses of the antiretroviral (ART) agent, and
optionally
the latency-reversing agent, are to be administered to the subject before
receiving said
combination.
In some embodiments, said combination is to be administered to the subject
until no cell
containing replication-competent proviral HIV DNA is detected in a blood
sample from
the subject.
DEFINITIONS
In the present invention, the following terms have the following meanings:
- "About" preceding a figure encompasses plus or minus 10%, or
less, of the value of
said figure. It is to be understood that the value to which the term "about"
refers is
itself also specifically, and preferably, disclosed.
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- As used herein, the term "adjuvant" refers to a compound or combination of
compounds that helps and enhances the pharmacological effect of a drug or a
vaccine,
or increases an immunogenic response.
- The term "administering" means either directly administering a compound or
composition of the present invention, or administering a prodrug, derivative
or analog
which will form an equivalent amount of the active compound or substance
within
the body. Exemplary routes of administration include, but are not limited to,
injection
(such as subcutaneous, intramuscular, intradermal, intraperitoneal, and
intravenous),
oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation
routes.
- The term "antigen" refers to a compound, composition, or substance that can
stimulate the production of antibodies or a T cell response in an animal,
including
compositions that are injected or absorbed into an animal. An antigen reacts
with the
products of specific humoral or cellular immunity, including those induced by
heterologous immunogens. The term "antigen" includes all related antigenic
epitopes. "Epitope" or "antigenic determinant" refers to a site on an antigen
to
which B and/or T cells respond. Epitopes can be formed both from contiguous
amino
acids or noncontiguous amino acids juxtaposed by tertiary folding of a
protein.
Epitopes formed from contiguous amino acids are typically retained on exposure
to
denaturing solvents whereas epitopes formed by tertiary folding are typically
lost on
treatment with denaturing solvents. An epitope typically includes at least 3,
and more
usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial
conformation.
Methods of determining spatial conformation of epitopes include, for example,
x-ray
crystallography and 2-dimensional nuclear magnetic resonance.
-
The term "decrease" refers to reducing the quality, amount, or strength
of something.
For example, a therapy (such as the methods provided herein) decreases the
infectious
load or titer of a pathogen such as HIV, or one or more symptoms associated
with
infection.
- The term "fragment" refers to a portion of a polypeptide that exhibits at
least one
useful epitope. The phrase "functional fragment(s) of a polypeptide" refers to
all
fragments of a polypeptide that retain an activity, or a measurable portion of
an
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activity, of the polypeptide from which the fragment is derived. Fragments,
for
example, can vary in size from a polypeptide fragment as small as an epitope
capable
of binding an antibody molecule to a large polypeptide capable of
participating in the
characteristic induction or programming of phenotypic changes within a cell.
5 An
epitope is a region of a polypeptide capable of binding an immunoglobulin
generated in response to contact with an antigen.
-
The term "immunogenic peptide" (or "antigenic peptide") refers to a
peptide which
comprises an allele-specific motif or other sequence, such as an N-terminal
repeat,
such that the peptide will bind an MHC molecule and induce a cytotoxic T
10
lymphocyte ("CTL") response, or a B cell response (for example antibody
production)
against the antigen from which the immunogenic peptide is derived. In one
embodiment, immunogenic peptides arc identified using sequence motifs or other
methods, such as neural net or polynomial determinations known in the art.
Typically,
algorithms are used to determine the "binding threshold" of peptides to select
those
with scores that give them a high probability of binding at a certain affinity
and will
be immunogenic. The algorithms are based either on the effects on MHC binding
of
a particular amino acid at a particular position, the effects on antibody
binding of a
particular amino acid at a particular position, or the effects on binding of a
particular
substitution in a motif-containing peptide. Within the context of an
immunogenic
peptide, a "conserved residue" is one which appears in a significantly higher
frequency than would be expected by random distribution at a particular
position in a
peptide.
-
The term "immunity" refers to the state of being able to mount a
protective response
upon exposure to an immunogenic agent. Protective responses can be
antibody-mediated or immune cell-mediated, and can be directed toward a
particular
pathogen or tumor antigen. Immunity can be acquired actively (such as by
exposure
to an immunogenic agent, either naturally or in a pharmaceutical composition)
or
passively (such as by administration of antibodies or in vitro stimulated and
expanded
T cells).
- As used herein, the term "alpha interferon" (IFN-a) or "interferon-alpha"
refers to
a family of more than 20 related but distinct members encoded by a cluster on
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chromosome 9 and all bind to the same IFN receptor. Among these, the IFN-a2
have
3 recombinant variants (a2a, a2b, a2c) depending upon the cells of origin and
the
IFN-a2b is the predominant variant in human genome. There is evidence though
that
each subtype has a different binding capacity to the IFNAR, modulating the
signaling
transduction events and the biological effects in the target cells.
- The term "type III interferon", also called interferon-lambda (IFN-X),
refers to
naturally occurring and/or recombinant cytokines of the type III interferon-
lambda
family. There are four IFN-X, members in humans, IFN-2l/IL-29, IFN-72/IL-28A,
IFN-X3/IL-28B,
- The term "beta interferon" (1FN-13) or "interferon-beta" refers to a family
of two
related but distinct members IFN-131 and IFN-I33. They both bind to the same
1FN
receptor, the 1FN-a receptor (IFNAR), which is a cell surface receptor complex
consisting of 2 chains: IFNAR1 and IFNAR2 (IFNAR1/IFNAR2 heterodimer).
Binding of IFN-13 to the IFNAR receptor triggers signaling transduction events
and
biological effects in the target cell.
- The term "isolated" or "non-naturally occurring" with reference to a
biological
component (such as a nucleic acid molecule, protein organelle or cells),
refers to a
biological component altered or removed from the natural state. For example, a
nucleic acid or a peptide naturally present in a living animal is not
"isolated," but the
same nucleic acid or peptide partially or completely separated from the
coexisting
materials of its natural state is "isolated". An isolated nucleic acid or
peptide can exist
in substantially purified form, or can exist in a non-native environment such
as, for
example, a host cell. Typically, a preparation of isolated nucleic acid or
peptide
contains the nucleic acid or peptide at least about 80% pure, at least about
85% pure,
at least about 90% pure, at least about 95% pure, greater than 95% pure,
greater than
about 96% pure, greater than about 97% pure, greater than about 98% pure, or
greater
than about 99% pure. Nucleic acids and proteins that are "non-naturally
occurring" or
have been "isolated" include nucleic acids and proteins purified by standard
purification methods. The term also embraces nucleic acids and proteins
prepared by
recombinant expression in a host cell as well as chemically synthesized
nucleic acids.
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An "isolated polypeptide" is one that has been identified and separated and/or
recovered from a component of its natural environment.
- The terms "subject", "individual," and "patient" are used interchangeably
herein,
and refer to an animal, for example a mammal, primate or human. and include
all
mammals, such as e.g. non-human primate, (particularly higher primates),
sheep, dog,
rodent, (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows,
horses.
In particular, these terms refer to a human, to whom treatment, including
prophylactic
treatment, with the combination according to the present invention, is
provided.
- The tem' "mutation" refers to any difference in a nucleic acid or
polypeptide
sequence from a normal, consensus or "wild type" sequence. A mutant is any
protein
or nucleic acid sequence comprising a mutation. In addition, a cell or an
organism
with a mutation may also be referred to as a mutant. Some types of coding
sequence
mutations include point mutations (differences in individual nucleotides or
amino
acids); silent mutations (differences in nucleotides that do not result in an
amino acid
changes); deletions (differences in which one or more nucleotides or amino
acids are
missing, up to and including a deletion of the entire coding sequence of a
gene);
frameshift mutations (differences in which deletion of a number of nucleotides
indivisible by 3 results in an alteration of the amino acid sequence. A
mutation that
results in a difference in an amino acid may also be called an amino acid
substitution
mutation. Amino acid substitution mutations may be described by the amino acid
change relative to wild type at a particular position in the amino acid
sequence.
- As used herein, the terms "prevent", "preventing" and "prevention" refer to
preventative measures, wherein the object is to reduce the chances that a
subject will
develop the pathologic condition or disorder over a given period of time. Such
a
reduction may be reflected, e.g., in a delayed onset of at least one symptom
of the
pathologic condition or disorder in the subject.
- The term "prophylactic" refers to a treatment administered to
a subject who does not
exhibit signs of a disease or exhibits only early signs for the purpose of
decreasing
the risk of developing pathology. In particular, a prophylactic treatment of a
HIV or
SIV infection in a subject refers to a treatment that allows the subject to
become an
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elite controller (EC) i.e. to have a relatively high CD4 + T cell count (such
as
e.g. superior to 500 CD4 + T cells per microliter) and/or to maintain
clinically
undetectable plasma HIV-1 RNA level (such as e.g. HIV RNA <50 copies/mL)
during
a prolonged period of time in the absence of any antiretroviral treatment
(ART).
- The term
"therapeutic" refers to a treatment administered to a subject who exhibit
early or established signs of a disease.
- The term "curative" refers to a treatment administered to a subject
suffering from a
disease for the purpose of curing the disease, i.e. of making any sign of the
disease
disappear or becoming undetectable.
- The terms "protein", "peptide", "polypeptide", and "amino acid sequence" are
used
interchangeably herein to refer to polymers of amino acid residues of any
length.
The polymer can be linear or branched, it may comprise modified amino acids or
amino acid analogs, and it may be interrupted by chemical moieties other than
amino
acids. The terms also encompass an amino acid polymer that has been modified
naturally or by intervention; for example disulfide bond formation,
glycosylation,
lipidation, acetylation, phosphorylation, or any other manipulation or
modification,
such as conjugation with a labeling or bioactive component.
-
The term "sample" or "biological sample" refers to a biological specimen
obtained
from a subject, such as a cell, fluid of tissue sample. In some cases,
biological samples
contain genomic DNA, RNA (including mRNA and microRNA), protein, or
combinations thereof. Examples of samples include, but are not limited to,
saliva,
blood, serum, urine, spinal fluid, tissue biopsy, surgical specimen, cells
(such as
PBMCs, white blood cells, lymphocytes, or other cells of the immune system)
and
autopsy material.
- As used herein, the term "treatment" refers to an intervention that
ameliorates a sign
or symptom of a disease or pathological condition. For example, in case of HIV
infection, HIV RNA (viral load) and CD4 T lymphocyte (CD4) cell count are the
two
surrogate markers of antiretro viral treatment (ART) responses and HIV disease
progression that have been used for decades to manage and monitor HIV
infection.
Thus, the efficacy of the treatment may be evaluated by the plasma viral RNA
load
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of a "treated" human before and after the treatment, if it is reduced by at
least about
10%, 20%, 30%, 40%, 50%, more preferably by at least about 70%, yet more
preferably by at least about 75% or 80% or 85% or 90% or 95% or 98% or 99%, or
even more (99.5%, 99.8%, 99.9%, 100%) the treatment is considered as
effective,
and/or by the monitoring of CD4 cell count before and after the treatment, if
the
absolute count of CD4 cell is increased by at least about 5%, 10%, 15%, 20%,
25% ,
more preferably by at least about 30%, yet more preferably by at least about
35% or
40% or 45% or 50% or 55% or 60% or 65%, or even more the treatment is
considered
as effective. As used herein, the terms "treatment", "treat" and "treating,"
with
reference to a disease, pathological condition or symptom, also refers to any
observable beneficial effect of the treatment. The beneficial effect can be
evidenced,
for example, by a delayed onset of clinical symptoms of the disease in a
susceptible
subject, a reduction in severity of some or all clinical symptoms of the
disease, a
slower progression of the disease, a reduction in the number of relapses of
the disease,
an improvement in the overall health or well-being of the subject, or by other
parameters well known in the art that are specific to the particular disease.
A therapeutic treatment is a treatment administered to a subject after signs
and
symptoms of the disease have developed. A prophylactic treatment is a
treatment
administered to a subject who does not exhibit signs of a disease or exhibits
only early
signs, for the purpose of decreasing the risk of developing pathology. Also, a
prophylactic treatment of a HIV or SIV infection in a subject refers to a
treatment that
allows the subject to become an elite controller (EC) i.e. to have a
relatively high
CD4 T cell count (such as e.g. superior to 500 CD4 + T cells per microliter)
and/or to
maintain clinically undetectable plasma HIV-1 RNA level (such as e.g. HIV RNA
<50 copies/mL) during a prolonged period of time in the absence of any
antiretroviral
treatment (ART). A prophylactic treatment is a treatment administered to a
subject
suffering from a disease for the purpose of curing the disease, i.e. of making
any sign
of the disease disappear or becoming undetectable.
- As used herein, the term "vaccine" refers to an immunogenic
product or composition
that can be administered to a mammal, such as a human, to confer immunity,
such as
passive or active immunity, to a disease or other pathological condition.
Vaccines can
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be used preventively or therapeutically, either prophylactically or
curatively.
Thus, vaccines can be used to reduce the likelihood of developing a disease
(such as
infection) or to reduce the severity of symptoms of a disease or condition,
limit the
progression of the disease or condition (such as infection), or limit the
recurrence of
5 a disease or condition.
- The term "virus" refers to microscopic infectious organism that reproduces
inside
living cells. A virus consists essentially of a core of nucleic acid (the
viral genome)
surrounded by a protein coat (capsid), and has the ability to replicate only
inside a
living cell. "Viral replication" is the production of additional virus
particles by the
10 occurrence of at least one viral life cycle. A virus may subvert the
host cells' normal
functions, causing the cell to behave in a manner determined by the virus.
For example, a viral infection may result in a cell producing a cytokine, or
responding
to a cytokine, when the uninfected cell does not normally do so. Particular
viral
species can alternatively enter into a "lysogenic" or "latent" infection.
15 In the establishment of latency, the viral genome is replicated, but
capsid proteins are
not produced and assembled into viral particles.
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DETAILED DESCRIPTION
The invention relates to a method for treating acquired immune deficiency
syndrome
(AIDS) in a subject in need thereof, comprising administering to the subject a
combination comprising:
i) a type III interferon blocking agent,
ii) an interferon-alpha (IFN-a) blocking agent,
iii) optionally, an interferon-beta (IFN-f3) blocking agent,
iv) an antiretroviral (ART) agent, and
v) optionally, a latency-reversing agent (LRA).
In one embodiment, the method comprises administering to the subject i) a type
III
interferon blocking agent, ii) an interferon-alpha (IFN-a) blocking agent, and
iv) an
antiretroviral (ART) agent.
In another embodiment, the method comprises administering to the subject i) a
type III
interferon blocking agent, ii) an interferon-alpha (IFN- a) blocking agent,
iii) an
interferon-beta (IFN-13) blocking agent, and iv) an antiretroviral (ART)
agent.
In another embodiment, the method comprises administering to the subject i) a
type III
interferon blocking agent, ii) an interferon-alpha (IFN-a) blocking agent,
iii) an
interferon-beta (IFNI.) blocking agent, iv) an antiretroviral (ART) agent, and
v)
optionally, a latency-reversing agent (LRA).
In some embodiments, the method is a method of prophylactic treatment.
In some embodiments, the method is a method of curative treatment.
The present invention further relates to a combination comprising:
i) a type 111 interferon blocking agent,
ii) an interferon-alpha (1FN-a) blocking agent,
iii) optionally, an interferon-beta (IFN-13) blocking agent,
iv) optionally, an antiretroviral (ART) agent, and
v) optionally, a latency-reversing agent (LRA).
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The present invention also relates to a combination for use as a medicament,
wherein said
combination comprises:
i) a type III interferon blocking agent,
ii) an interferon-alpha (IFN-a) blocking agent,
iii) optionally, an interferon-beta (IFN-I3) blocking agent,
iv) an antiretroviral (ART) agent, and
v) optionally, a latency-reversing agent (LRA).
The present invention also relates to a combination for use in the treatment
of acquired
immune deficiency syndrome (AIDS) in a subject in need thereof, wherein said
combination comprises:
i) a type III interferon blocking agent,
ii) an interferon-alpha (IFN-ct) blocking agent,
iii) optionally, an interferon-beta (IFN-I3) blocking agent,
iv) an antiretroviral (ART) agent, and
v) optionally, a latency-reversing agent (LRA).
In one embodiment, the combination for use comprises i) a type III interferon
blocking
agent, ii) an interferon-alpha (IFN-a) blocking agent, and iv) an
antiretroviral (ART)
agent.
In one embodiment, the combination for use comprises i) a type III interferon
blocking
agent, ii) an interferon-alpha (IFN-a) blocking agent, iii) an interferon-beta
(IFN-I3)
blocking agent, and iv) an antiretroviral (ART) agent.
In one embodiment, the combination for use comprises i) a type III interferon
blocking
agent, ii) an interferon-alpha (IFN-a) blocking agent, iii) an interferon-beta
(IFN-I3)
blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a
latency-reversing
agent (LRA).
The present invention further relates to a kit-of-parts comprising:
i) a type III interferon blocking agent,
ii) an interferon-alpha (IFN-a) blocking agent,
iii) optionally, an interferon-beta (IFN-I3) blocking agent,
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iv) optionally, an antiretroviral (ART) agent, and
v) optionally, a latency-reversing agent (LRA).
The present invention further relates to a kit-of-parts for use as a
medicament, wherein
said kit-of-parts comprises:
i) a type III interferon blocking agent,
ii) an interferon-alpha (IFN-a) blocking agent,
iii) optionally, an interferon-beta (IFN-I3) blocking agent,
iv) optionally, an antiretroviral (ART) agent, and
v) optionally, a latency-reversing agent (LRA).
The present invention also relates to a kit-of-parts for use in the treatment
of acquired
immune deficiency syndrome (AIDS) in a subject in need thereof, wherein
said kit-of-parts comprises:
i) a type III interferon blocking agent,
ii) an interferon-alpha (IFN-a) blocking agent,
iii) optionally, an interferon-beta (IFN-I3) blocking agent,
iv) an antiretroviral (ART) agent, and
v) optionally, a latency-reversing agent (LRA).
In one embodiment, the kit-of-parts for use comprises i) a type III interferon
blocking
agent, ii) an interferon-alpha (IFN-i) blocking agent, and iv) an
antiretroviral (ART)
agent.
In one embodiment, the kit-of-parts for use comprises i) a type III interferon
blocking
agent, ii) an interferon-alpha (IFN-a) blocking agent, iii) an interferon-beta
(IFN-I3)
blocking agent, and iv) an antiretroviral (ART) agent.
In one embodiment, the kit-of-parts for use comprises i) a type III interferon
blocking
agent, ii) an interferon-alpha (IFN-a) blocking agent, iii) an interferon-beta
(IFN-I3)
blocking agent, iv) an antiretroviral (ART) agent, and v) optionally, a
latency-reversing
agent (LRA).
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According to the present invention, the combination or kit-of-parts as
described herein is
for use in the treatment of acquired immune deficiency syndrome (AIDS) in a
subject in
need thereof.
In one embodiment, the subject is infected with a human immunodeficiency virus
(HIV),
or a simian immunodeficiency virus (S IV).
Thus, the combination or kit-of-parts as described herein is also for use in
the treatment
of a human immunodeficiency virus (HIV) infection or a simian immunodeficiency
virus
(SIV) infection.
Due to the great variability in the HIV genome, which results from mutation,
recombination, insertion and/or deletion, HIV has been classified in groups,
subgroups,
types, subtypes and genotypes. There are two major HIV groups (HIV-1 and HIV-
2) and
many subgroups because the HIV genome mutates constantly. The major difference
between the groups and subgroups is associated with the viral envelope. HIV-1
is
classified into a main group (M), said group M being divided into least nine
genetically
distinct subtypes. These are subtypes A, B, C, D, F, G, H, J and K. Many other
subtypes
resulting from in vivo recombination of the previous ones also exist (e.g.,
CRF).
In one embodiment, the HIV antigen is related to a specific HIV group,
subgroup, type,
subtype or to a combination of several subtypes.
In one embodiment, the HIV virus is HIV-1 or HIV-2, preferably HIV-1.
In one embodiment, the subject is infected with an HIV-1 strain or an HIV-2
strain.
Thus, the combination or kit-of-parts as described herein is also for use in
the treatment
of an HIV-1 strain or an HIV-2 strain.
In one embodiment, the HIV-1 virus is from group M and preferably subtype B
(HXB2).
In one embodiment, the subject is a mammal, a primate, preferably a human.
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In an embodiment, the combination or kit-of-parts as described herein is for
use in the
preventive or the prophylactic treatment of acquired immune deficiency
syndrome
(AIDS) in a subject in need thereof.
In another embodiment, the combination or kit-of-parts as described herein is
for use in
5 the curative treatment of acquired immune deficiency syndrome (AIDS) in a
subject in
need thereof.
In some embodiments, the subject has already received at least one dose of at
least one
antiretroviral (ART) agent, or is under antiretroviral therapy or under
combined
antiretroviral therapy (cART) comprising at least one antiretroviral (ART)
agent, before
10 being administered the combination of the invention.
As used herein, the term "alpha interferon" (IFN-a) or "interferon-alpha"
refers to a
family of more than 20 related but distinct members encoded by a cluster on
chromosome
9 and all bind to the same IFN receptor. Among these, the IFN-a2 have 3
recombinant
variants (ot2a, nib, a2c) depending upon the cells of origin and the IFN-a2b
is the
15 predominant variant in human genome. There is evidence though that each
subtype has a
different binding capacity to the IFNAR, modulating the signaling transduction
events
and the biological effects in the target cells.
In one embodiment, the interferon-alpha blocking agent described herein is an
agent
neutralizing circulating IFN-a and/or an agent blocking IFN-a signaling,
and/or an agent
20 depleting IFN-a producing cells, and/or an agent blocking IFN-a
production.
In one embodiment, the interferon-alpha blocking agent described herein
comprises at
least one agent selected from: an agent neutralizing circulating IFN-ct and/or
an agent
blocking IFN-a signaling, and/or an agent depleting IFN-a producing cells,
and/or an
agent blocking IFN-a production.
In one embodiment, the agent neutralizing circulating IFN-a and/or the agent
blocking
IFN-a signaling, and/or the agent depleting IFN-a producing cells, and/or the
agent
blocking IFN-a production is/are an IFN-a antagonist.
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In some embodiments, the interferon-alpha blocking agent is selected from the
group
consisting of : an agent neutralizing circulating alpha interferon, an agent
blocking
interferon-alpha signaling, an agent depleting IFN-a, producing cells, and/or
an agent
blocking IFN-a production, wherein the agent neutralizing circulating alpha
interferon is
selected from the group comprising active anti-IFN-a vaccine including IFN-a-
kinoid,
1FN-a DNA-based or IFN-a RNA-based vaccine, or passive anti-IFN-a, vaccine
including
anti-IFN-a antibodies or anti-IFN-a hyper-immune serum, wherein the blocking
agent of
interferon-alpha signaling is selected from the group consisting of an active
anti-IFNAR1
or anti-IFNAR2 vaccine, such as an IFNAR1 or IFNAR2 DNA-based or an IFNAR1 or
IFNAR2 RNA-based vaccine, a passive anti-IFNAR1 or anti-IFNAR2 vaccine, such
as
an anti-type I interferon R1 or R2 antibody, and an IFN-a endogenous
regulator, such as
SOSC1 or an aryl hydrocarbon receptor, wherein the agent depleting IFN-oc
producing
cells is an agent depleting plasmacytoid dendritic cells (pDCs), and wherein
the agent
blocking IFN-a production is an agent blocking the production of IFN-a by
pDCs.
In some embodiments, the interferon-alpha blocking agent is an agent
neutralizing
circulating alpha interferon selected from the group consisting of active
anti-1FN-a vaccine including 114N-a-kinoid, IFN-a, DNA-based or 114N-a RNA-
based
vaccine, or passive anti-IFN-a vaccine including anti-IFN-a antibodies or anti-
IFN-a
hyper-immune serum, and the blocking agent of interferon-alpha signaling is
selected
from the group consisting of an active anti-IFNAR1 or anti-IFNAR2 vaccine,
such as an
IFNAR1 or IFNAR2 DNA-based or an IFNAR1 or IFNAR2 RNA-based vaccine, a
passive anti-IFNAR1 or anti-IFNAR2 vaccine, such as an anti-type 1 interferon
R1 or R2
antibody, and an IFN-a endogenous regulator, such as SOSC1 or an aryl
hydrocarbon
receptor.
As used herein, the term "alpha interferon antagonist" refers to a substance
which
interferes with or inhibits the TEN-a biological activity. "IFN-oc biological
activity" as
used herein refers to any activity occurring as a result of 1FN-a binding to
its receptor
IFNAR (IFNAR1/IFNAR2 heterodimer). Such binding can, for example, activate the
JAK-STAT signaling cascade, and trigger tyrosine phosphorylation of a number
of
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proteins including JAKs, TYK2, STAT proteins. Thus, the blocking agent of
interferon-
alpha signaling can neutralize the fixation of the INF-a to its receptor
and/or block the
signaling cascade induced by the binding of IFN-a to its receptor. In some
embodiments,
the IFN-a antagonist is selected from the group of active anti-TEN-a vaccine
(e.g., TFN-
a-kinoid, IFN-a DNA-based or IFN-a RNA-based vaccine) or passive anti-IFN-a
vaccine
(e.g., anti-IFN-a antibody or anti-IFN- a hyper-immune serum). See for example
Noel et
al. (2018). Cytokine Growth Factor Rev 40:99-112.
In one embodiment, the agent neutralizing circulating IFN-ot is a passive anti-
IFN-a
vaccine, such as an anti-IFN-a antibody or an anti-IFN-a hyper-immune serum.
In one embodiment, the agent neutralizing circulating IFN-a is an anti-IFN-a
antibody,
preferably a neutralizing antibody. The anti-TEN-a antibody may he a
monoclonal or a
polyclonal antibody, and is preferably a monoclonal antibody.
Examples of anti-IFN-a antibodies include, without limitation, Sifalimumab,
Rontalizumab, MMHA-1 clone, MMHA-2 clone, MMHA-6 clone, MMHA-8 clone,
MMHA-9 clone, MMHA-11 clone, MMHA-13 clone and MMHA-17 clone.
In one embodiment, the agent neutralizing circulating IFN-a is an anti-IFN-a
hyper-immune serum.
In one embodiment, the agent neutralizing circulating IFN-a described herein
is an
IFN-a ligand inhibitor.
In one embodiment, the agent neutralizing circulating IFN-a is a soluble
receptor that
binds lFN-a.
In another embodiment, the agent neutralizing circulating IFN-a is an active
anti-IFN-a
vaccine.
An "active anti-IFN-a vaccine" designates a compound or composition that is
capable of
inducing the production of anti-IFN-ct antibodies. For instance, an -active
anti-IFN-a
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vaccine" designates a compound or composition which, upon administration to a
subject,
is capable of inducing the production of anti-IFN-a auto-antibodies by said
subject.
The active ingredient of the active anti-IFN-a vaccine may be a polypeptide, a
protein, a
DNA or an RNA molecule.
For instance, the active ingredient of the active anti-IFN-a vaccine is an IFN-
a-kinoid. A
kinoid is an inactivated and/or non-toxic IFN derivative with immunogenic
properties.
Usually, it is in the form of a heterocomplex obtained by chemical binding of
the IFN
derivative to a carrier.
The kinoid may be used as an immunogen capable of inducing high affinity auto-
antibodies against a given IFN. Immunization with kinoids thus induce high
titers of
neutralizing antibodies directed against the corresponding IFN.
In one embodiment, the agent neutralizing circulating IFN-a described herein
is an IFN-
a¨kinoid, such as, for example, Antiferon .
The active ingredient of the active anti-IFN-a vaccine may also be a DNA
molecule, for
instance part(s) of or full-length IFN-a DNA, or an RNA molecule, for instance
part(s)
of or full-length IFN-a RNA.
The active anti-IFN-a vaccine may induce the production of antibodies that
binds to one
IFN-ot subtype or to several IFN-a subtypes. For instance, the RNA molecule
used in the
IFN-a RNA-based vaccine may be specific of one IFN-a subtype. Alternatively,
the
active anti-IFN-a vaccine may comprise at least two RNA molecules
corresponding to at
least two IFN-a subtypes.
In another embodiment, the interferon-alpha blocking agent is an agent
blocking IFN-
a signaling.
In one embodiment, the agent blocking IFN-a signaling is an agent that
antagonizes the
type I IFN signaling pathway.
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In one embodiment, the agent blocking IFN-a signaling described herein is an
IFNAR
antagonist.
In one embodiment, the agent blocking IFN-a signaling is an IFNAR1 antagonist.
In another embodiment, the agent blocking IFN-a signaling is an IFNAR2
antagonist.
In one embodiment, the agent blocking IFN-a signaling is a passive anti-IFNAR1
or
anti-IFNAR2 vaccine, such as an anti-type I interferon R1 or R2 antibody.
In one embodiment, the agent blocking IFN-a signaling is an antibody that
binds to
IFNAR1 or IFNAR2.
In another embodiment, the agent blocking TEN-a signaling is an active anti-
IFNAR1 or
anti-IFNAR2 vaccine, such as an IFNAR1 or IFNAR2 DNA-based or an IFNAR1 or
IFNAR2 RNA-based vaccine.
An "active anti-IFNAR1 or anti-IFNAR2 vaccine" designates a compound or
composition that is capable of inducing the production of anti-IFNAR1 or anti-
IFNAR2
antibodies. For instance, an "active anti-IFNAR1 or anti-IFNAR2 vaccine"
designates a
compound or composition which, upon administration to a subject, is capable of
inducing
the production of anti-IFNAR1 or anti-IFNAR2 auto-antibodies by said subject.
The active ingredient of the active anti-IFNAR1 or anti-IFNAR2 vaccine may be
a DNA
molecule, for instance part(s) of or full-length IFNAR1 or IFNAR2 DNA, or an
RNA
molecule, for instance part(s) of or full-length IFNAR1 or IFNAR2 RNA.
In one embodiment, the agent blocking IFN-a signaling can be an inhibitor of
type I IFN
signaling pathway. Type I IFN signaling pathway inhibitors are well known in
the art and
include, without limitation, JAK1/2/3 inhibitors and STAT inhibitors.
Accordingly, in
one embodiment, the agent blocking IFN-cc signaling is selected from JAK1/2/3
inhibitors, STAT inhibitors, and Tyrosine Kinase 2 (TYK2) inhibitors. Non-
limiting
examples of JAK1/2/3 inhibitors include Ruxolitinib, Tofacitinib and
Baricitinib.
Non-limiting examples of TYK2 inhibitors include the BMS-986165 inhibitor.
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In one embodiment, the agent blocking IFN-a signaling can be an endogenous
negative
regulator of type I IFN signaling pathway. Endogenous negative regulators are
well
known in the art and include, without limitation, SOCS1/3, FOX03, Aryl
hydrocarbon
Receptor (AhR) or other negative regulators. Accordingly, in one embodiment,
the agent
5 blocking interferon signaling is selected from SOCS1/3, FOX03 or Aryl
hydrocarbon
Receptor (AhR).
In one embodiment, the agent blocking IFN-a signaling is a PASylated
antagonist.
PASylated antagonist of type 1 1FN arc known in the art, see for example
Nganou-Makamdop et at. (2018). PLoS Pathog 14(8): e1007246.
10 In one embodiment, the IFN-a antagonist described herein is an agent
depleting
IFN-cc producing cells.
As used herein, the term "IFN-a producing cells" refers to any cell that
produce IFN-a.
In particular, it is well known in the art that the plasmacytoid dendritic
cells (pDCs) are
the main producer of TEN-a. Thus, in one embodiment, the agent depleting
15 IFN-a producing cells depletes pDCs.
In one embodiment, the agent depleting IFN-a producing cells is an antibody.
In one embodiment, the antibody depletes pDCs. such as, for example, an anti-
CD123
antibody (i.e., anti-IL-3RA).
In one embodiment, the IFN-a antagonist described herein is an agent that
blocks the
20 production of TEN-a.
In one embodiment, the agent that blocks the production of the IFN-a is an
antibody.
In one embodiment, the antibody blocks the production of IFN-a by pDCs. Said
antibody
can be, for example, an anti-BDCA2 (Blood DC Antigen 2) antibody.
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In some embodiments, the interferon-alpha blocking agent is selected from the
group
consisting of:
- an anti-IFN-a antibody, preferably Sifalimumab, Rontalizumab, MMHA-1 clone,
MMHA-2 clone, MMHA-6 clone, MMHA-8 clone, MMHA-9 clone,
MMHA-11 clone, MMHA-13 clone or MMHA-17 clone,
- an anti-IFN-a hyper-immune serum,
- an IFN-ct¨kinoid, such as e.g. Antiferone,
- an IFN-ct DNA-based or an IFN-a RNA-based vaccine,
- a soluble receptor that binds IFN-a,
- an IFNAR1 or IFNAR2 antagonist, preferably an antibody that binds to IFNAR1
or
IFNAR2,
- an IFNAR1 or IFNAR2 DNA-based or an IFNAR1 or IFNAR2 RNA-based vaccine,
- a type I IFN signaling pathway inhibitors selected from a STAT inhibitor,
a JAK1/2/3
inhibitor, such as e.g. Ruxolitinib, Tofacitinib or Baricitinib, and a TYK2
inhibitor,
such as e.g. BMS-986165,
- an endogenous negative regulator of type I IFN signaling pathway selected
from
SOCS1/3, FOX03, Aryl hydrocarbon Receptor (AhR) or another negative regulator,
- a PAS ylated antagonist,
- an antibody depleting pDCs, preferably an anti-CD123 (i.e. anti-IL-3RA)
antibody,
- an antibody blocking the production of IFN-a by pDCs, preferably an anti-
BDCA2
(Blood DC Antigen 2) antibody.
In one embodiment, the interferon-alpha blocking agent is an anti-IFN-a
antibody,
preferably a monoclonal antibody, more preferably a neutralizing antibody.
As used herein, the term "type III interferon", also called interferon-lambda
(IFN-2),
refers to naturally occurring and/or recombinant cytokines of the type III
interferon-lambda family. There are four IFN-X members in humans, IFN-kJ/IL-
29,
IFN-X2/IL-28A, IFN-23/IL-28B, IFN-X4.
In one embodiment, the type III interferon is IFN-X.
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In one embodiment, the IFN-X, refers to at least one IFN-X, subtype, i.e. IFN-
2d, IFN-X2
IFN-X3,
In one embodiment, the human IFN-71 has the following accession number
NP_742152.1. In one embodiment, the human IFN-22 has the following accession
number NP_742150.1. In one embodiment, the human IFN-23 has the following
accession numbers NP_001333866.1 (isoform 1) or NP_742151.2 (isoform 2). In
one
embodiment, the human IFN-2.4 has the following accession number
NP_001263183.2.
In some embodiments, the type III interferon to be blocked is a mucosal type
III interferon
or a mucosal IFN-X.
In one embodiment, the interferon-lambda blocking agent described herein is an
agent
neutralizing circulating IFN-X and/or an agent blocking IFN-X signaling.
Preferably, the interferon-lambda blocking agent is an agent neutralizing
mucosal IFN-
X and/or an agent blocking mucosal IFN-X signaling.
In one embodiment, the agent neutralizing circulating IFN-X and/or the agent
blocking
IFN-X, signaling is/are an IFN-X, antagonist.
In some embodiments, the at least one type III interferon blocking agent is:
- an agent neutralizing circulating IFN-X, selected from the group
comprising an active
anti-IFN-X vaccine, such as an IFN-X-kinoid,
DNA-based or IFN-X RNA-based
vaccine, and a passive anti-IFN-X vaccine, such as an anti-1FN-X antibody or
an anti-IFN-
X hyper-immune serum, or
- an agent blocking type III interferon signaling selected from the group
consisting of an
active anti-IFNLR1 vaccine, such as an IFNLR1 DNA-based or IFNLR1 RNA-based
vaccine, and a passive anti-IFNLR1 vaccine, such as an antibody that binds to
the
IFNLR1 receptor.
As used herein, the term "lambda interferon antagonist" refers to a substance
which
interferes with or inhibits the IFN-X, biological activity. "IFN-A, biological
activity" as
used herein refers to any activity occurring as a result of IFN-X, binding to
its receptor
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IFNLR (IFNLR1/ILlOR2 heterodimer). Thus, the signaling blocking agent of
interferon
can neutralize the fixation of the INF-X to its receptor and/or block the
signaling cascade
induced by the binding of IFN-X to its receptor. In some embodiments, the IFN-
X
antagonist is selected from the group of active anti-IFN-X vaccine
(e.g., IFN-X-kinoid, IFN-X, DNA-based or IFN-X RNA-based vaccine) or passive
anti-
IFN-X, vaccine (e.g., anti-IFN-X antibody or anti-IFN-X hyper-immune serum).
In one embodiment, the agent neutralizing circulating IFN-X is a passive anti-
IFN-X
vaccine, such as an anti-IFN-X, antibody or an anti-IFN-X, hyper-immune serum.
In one embodiment, the agent neutralizing circulating IFN-X is an anti-IFN-X.
antibody,
preferably a neutralizing antibody.
The anti-interferon-lambda antibody may be a monoclonal or a polyclonal
antibody, and
is preferably a monoclonal antibody.
Non limiting examples of neutralizing anti-interferon-lambda antibodies
include:
- the monoclonal anti-IL-29 (IFN-X1) antibody clone 6A11
(Invivogen),
- the monoclonal anti-human IL-29 (IFN-X1) antibody clone #247801 (R&D
systems),
- the monoclonal anti-IL-28A (IFN-X2) antibody clone 21C3
(Invivogen),
- the monoclonal anti-IL-28 A (IFN-22) antibody clone MMHL-2 (PBL assay
sciences),
- the monoclonal anti-human IL-28A (IFN-X2) antibody Clone #248526
(R&D systems),
- the polyclonal anti-human IL-28A (IFN-k2) antibody (R&D systems),
- the monoclonal anti 1L-28B (1FN-23) antibody clone 18F4 (Invivogen), and
- the monoclonal anti-IL-28B (IFN-X3) antibody clone MMHL-3
(PBL assay
sciences).
In one embodiment, the agent neutralizing circulating IFN-X., is an anti-IFN-
X,
hyper-immune serum.
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In one embodiment, the agent neutralizing circulating IFN-X described herein
is an
ligand inhibitor.
In one embodiment, the agent neutralizing circulating IFN-X is a soluble
receptor that
binds IFN-X.
In another embodiment, the agent neutralizing circulating IFN-X, is an active
anti-IFN-X,
vaccine.
An -active anti-IFN-X vaccine" designates a compound or composition that is
capable of
inducing the production of anti-IFN-X antibodies. For instance, an "active
anti-IFN-k
vaccine" designates a compound or composition which, upon administration to a
subject,
is capable of inducing the production of anti-IFN-A auto-antibodies by said
subject.
The active ingredient of the active anti-IFN-2 vaccine may be a polypeptide, a
protein, a
DNA or an RNA molecule.
For instance, the active ingredient of the active anti-1FN-2 vaccine is an 1FN-
2-kinoid. A
kinoid is an inactivated and/or non-toxic IFN derivative with immunogenic
properties.
Usually, it is in the form of a heterocomplex obtained by chemical binding of
the IFN
derivative to a carrier.
The kinoid may be used as an immunogen capable of inducing high affinity auto-
antibodies against a given IFN. Immunization with kinoids thus induce high
titers of
neutralizing antibodies directed against the corresponding IFN.
The active ingredient of the active anti-1FN-2. vaccine may also be a DNA
molecule, for
instance part(s) of or full-length 1FN-2 DNA, or an RNA molecule, for instance
part(s) of
or full-length IFN-2 RNA.
The active anti-IFN-X, vaccine may induce the production of antibodies that
binds to one
IFN-X, subtype or to several IFN-X subtypes selected from the group comprising
IFN-2J,
IFN-22, IFN-23 and IFN-24. For instance, the RNA molecule used in the IFN-X
RNA-
based vaccine may be specific of one IFN-A subtype, i.e. specific of IFN-2.,1,
of IFN-22,
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of IFN-23 or of IFN-X4. Alternatively, the active anti-IFN-X vaccine may
comprise at
least two RNA molecules corresponding to at least two IFN-X subtypes selected
from the
group comprising IFN-Xl, 1FN-22, 1FN-23 and 1FN-k4.
In another embodiment, the type III interferon blocking agent is an agent
blocking type III
5 interferon signaling.
In one embodiment, the agent blocking IFN-X signaling is an agent that
antagonizes the
type III IFN signaling pathway.
In one embodiment, the agent blocking IFN-X, signaling described herein is an
IFNLR
antagonist.
10 In one embodiment, the agent blocking IFN-X signaling is an IFNLR1
antagonist.
In another embodiment, the agent blocking IFN-X signaling is an IL10R2
antagonist.
In one embodiment, the agent blocking type 111 interferon signaling is a
passive anti-
1FNLR1 vaccine, such as an antibody that binds to the IFNLR1 receptor.
In one embodiment, the agent blocking type III interferon signaling is an
antibody.
15 Such antibody can block or inhibit the biological effects of type III
interferon and/or block
or inhibit the type III interferon signaling pathway. For example, such
antibody may bind
to an epitope on the interferon-lambda receptor, impeding the binding of
interferon-
lambda to its receptor and thus the receptor signaling subsequent activation.
The
heterodimeric receptor complex of interferon-lambda (IFNLR) comprises IFNLR1
20 (1FNLRA, 1L-28RA), and ILl0R2 (1L-10RB). IFNLR1 confers ligand specificity
and
enables receptor assembly, while IL 10R2 is shared with 1L-10 family members
and is
required for signaling.
Thus, in one embodiment, the agent blocking type III interferon signaling is
an antibody
that binds to IFNLR1 or to IL10R2. In one embodiment, the agent blocking type
III
25 interferon signaling is an antibody that binds to the IFNLR1 receptor.
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Non-limiting examples of antibodies that bind to the IFNLR1 receptor include
the clone
MMHLR-1 (PM assay sciences) and the MHLICR2A1 antibody (Creative Biolabs).
In another embodiment, the agent blocking type III interferon signaling is an
active anti-
IFNLR1 vaccine, such as an IFNLR1 DNA-based or IFNLR1 RNA-based vaccine.
An "active anti-IFNLR1 vaccine" designates a compound or composition that is
capable
of inducing the production of anti-IFNLR1 antibodies. For instance, an "active
anti-
IFNLR1 vaccine" designates a compound or composition which, upon
administration to
a subject, is capable of inducing the production of anti-IFNLR1 auto-
antibodies by said
subject.
The active ingredient of the active anti-IFNLR1 vaccine may be a DNA molecule,
for
instance part(s) of or full-length IFNLR1 DNA, or an RNA molecule, for
instance part(s)
of or full-length IFNLR1 RNA.
In another embodiment, the type III interferon blocking agent is a small
chemical
molecule entity (such as, for example, a chemical entity with a molecular
weight less than
900 Daltons). Methods of screening chemical libraries to identify small
chemical
molecule entities which may be potential drug candidates are known in the art.
For example, a chemical library may be tested in a ligand-receptor binding
assay.
For instance, the small chemical molecule may bind and block the IFNLR1
receptor.
In some embodiments, the type III interferon blocking agent is selected from
the group
consisting of:
- an anti-IFN-X antibody, preferably clone 6A11, clone #247801. clone 21C3,
clone
MMHL-2, clone #248526, clone 18F4, or clone MMHL-3.
- an anti-IFN-X hyper-immune serum,
- an IFN-X¨kinoid,
- an IFN-X DNA-based or an IFN-X RNA-based vaccine,
- a soluble receptor that binds IFN-X,
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- an IFNLR1 or IL10R2 antagonist, preferably an antibody that binds to IFNLR1
or
ILlOR2, more preferably an antibody that binds to IFNLR1 such as clone MMHLR-
1 or the MHLICR2A1 antibody,
- an 1FNLR1 DNA-based or IFNLR1 RNA-based vaccine, and
- a small chemical molecule that binds to IFNLR1.
As used herein, the term "beta interferon" (IFN-I3) or -interferon-beta"
refers to a
family of two related but distinct members IFN-(31 and IFN-I33. They both bind
to the
same IFN receptor, the IFN-a receptor (IFNAR), which is a cell surface
receptor complex
consisting of 2 chains: IFNAR1 and IFNAR2 (IFNAR1/IFNAR2 heterodimer).
Binding of IFN-I3 to the IFNAR receptor triggers signaling transduction events
and
biological effects in the target cell.
In one embodiment, the interferon-beta blocking agent described herein is an
agent
neutralizing circulating IFN-f3 and/or an agent blocking IFN-I3 signaling.
In one embodiment, the interferon-beta blocking agent described herein
comprises at least
one agent selected from an agent neutralizing circulating IFNI:3 and an agent
blocking
IFNA3 signaling.
In one embodiment, the agent neutralizing circulating IFN-13 and/or the agent
blocking
IFN-f3 signaling is/are an IFN-I3 antagonist.
As used herein, the term "beta interferon antagonist" refers to a substance
which
interferes with or inhibits the IFN-I3 biological activity. " IFN-p biological
activity" as
used herein refers to any activity occurring as a result of IFN-13 binding to
its receptor
IFNAR (IFNAR1/IFNAR2 heterodimer). Such binding can, for example, activate the
JAK-STAT signaling cascade, and trigger tyrosine phosphorylation of a number
of
proteins including JAKs, TYK2, STAT proteins. Thus, the signaling blocking
agent of
interferon can neutralize the fixation of the IFN-I3 to its receptor and/or
block the
signaling cascade induced by the binding of IFN-I3 to its receptor. In some
embodiments,
the IFN-I3 antagonist is selected from the group of active anti-IFN-I3 vaccine
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(e.g., IFN-P-kinoid, IFN-P DNA-based or IFN-P RNA-based vaccine) or passive
anti-
IFN-P vaccine (e.g., anti-IFN-p antibody or anti-IFN-p hyper-immune serum).
In one embodiment, the agent neutralizing circulating IFN-I3 is a passive anti-
IFN-I3
vaccine, such as an anti-IFN-I3 antibody or an anti-IFN-P hyper-immune serum.
In one embodiment, the interferon-beta blocking agent is an agent neutralizing
circulating
IFN-p, wherein the agent neutralizing circulating IFN-I3 is an anti-IFN-p
antibody or
anti-IFN-P hyper-immune serum.
In one embodiment, the agent neutralizing circulating IFN-I3 is an anti-IFN-I3
antibody,
preferably a neutralizing antibody. The anti-IFN-P antibody may be a
monoclonal or a
polyclonal antibody, and is preferably a monoclonal antibody.
Non-limiting examples of anti-IFN-p antibodies include:
- the neutralizing monoclonal antibody against human TEN-beta, clone 10B10
(Invivogen)
- the polyclonal anti-human TEN-beta antibodies (R&D systems)
- the monoclonal anti-human TEN-beta antibodies clone #76703, clone #MMHB-3
and
clone #937912 (R&D systems)
- the neutralizing polyclonal anti-human IFN-beta goat IgG (PBL
assay sciences).
In one embodiment, the agent neutralizing circulating IFN-I3 is an anti-IFN-13
hyper-immune scrum.
In one embodiment, the agent neutralizing circulating IFN-P described herein
is an
1FN-p ligand inhibitor.
In one embodiment, the agent neutralizing circulating IFN-p is a soluble
receptor that
binds 1FN-P.
In another embodiment, the interferon-beta blocking agent is an active anti-
IFN-p
vaccine.
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An "active anti-IFN-r3 vaccine" designates a compound or composition that is
capable of
inducing the production of anti-IFN-f3 antibodies. For instance, an "active
anti-IFN-I3
vaccine" designates a compound or composition which, upon administration to a
subject,
is capable of inducing the production of anti-IFN-r3 auto-antibodies by said
subject.
The active ingredient of the active anti-IFN- P vaccine may be a polypeptide,
a protein, a
DNA or an RNA molecule.
For instance, the active ingredient of the active anti-IFN-I3 vaccine is an
IFN--kinoid. A
kinoid is an inactivated and/or non-toxic IFN derivative with immunogenic
properties.
Usually, it is in the form of a heterocomplex obtained by chemical binding of
the IFN
derivative to a carrier.
The kinoid may be used as an immunogen capable of inducing high affinity auto-
antibodies against a given IFN. Immunization with kinoids thus induce high
titers of
neutralizing antibodies directed against the corresponding IFN.
The active ingredient of the active anti-IFN-13 vaccine may also be a DNA
molecule, for
instance part(s) of or full-length IFN-f3 DNA, or an RNA molecule, for
instance part(s)
of or full-length IFN-r3 RNA.
In one embodiment, the interferon-beta blocking agent is an agent blocking
IFN-f3 signaling, wherein the blocking agent of IFN-13 signaling is selected
from the group
consisting of anti-type I interferon R1 or R2 antibodies, SOSC1 and aryl
hydrocarbon
receptors.
In one embodiment, the agent blocking IFN-f3 signaling described herein is an
IFNAR
antagonist. In one embodiment, the agent blocking IFN-I3 signaling is an
IFNAR1
antagonist. In another embodiment, the agent blocking IFN-13 signaling is an
IFNAR2
antagonist.
In one embodiment, the agent blocking IFN-I3 signaling is an antibody that
binds to
IFNAR1 or IFNAR2.
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In one embodiment, the agent blocking IFN-I3 signaling is an agent that
antagonizes the
type I IFN signaling pathway.
In one embodiment, the agent blocking IFN-13 signaling can be an inhibitor of
type I IFN
signaling pathway. Type I IFN signaling pathway inhibitors are well known in
the art and
5 include, without limitation, JAK1/2/3 inhibitors and STAT inhibitors.
Accordingly, in
one embodiment, the agent blocking IFN-11 signaling is selected from JAK1/2/3
inhibitors, STAT inhibitors, and Tyrosine Kinase 2 (TYK2) inhibitors. Non-
limiting
examples of JAK1/2/3 inhibitors include Ruxolitinib, Tofacitinib and
Baricitinib.
Non-limiting examples of TYK2 inhibitors include the BMS-986165 inhibitor.
10 In one embodiment, the agent blocking IFN-111 signaling can be an
endogenous negative
regulator of type I IFN signaling pathway. Endogenous negative regulators are
well
known in the art and include, without limitation, SOCS1/3, FOX03, Aryl
hydrocarbon
Receptor (AhR) or other negative regulators. Accordingly, in one embodiment,
the agent
blocking interferon signaling is selected from SOCS1/3, FOX03 or Aryl
hydrocarbon
15 Receptor (AhR).
In one embodiment, the agent blocking IFN-(3 signaling is a PASylated
antagonist.
PASylated antagonist of type I IFN are known in the art, see for example
Nganou-Makamdop et al. (2018). PLoS Pathog 14(8): e1007246.
As used herein, the term "antiretroviral therapy" or "ART" or "highly active
20 antiretroviral therapy" or "HAART" refers to any combination of
antiretroviral (ARV)
drugs to maximally suppress the HIV virus (e.g., reduce viral load reduce HIV
multiplication...), and stop the progression of HIV disease. There are several
classes of
HIV drug, such as, for example, non-nucleoside reverse transcriptase
inhibitors
(NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), post-attachment
25 inhibitors (or entry inhibitors), protease inhibitors (PIs), CCR5
antagonists, integrase
strand transfer inhibitors (INSTIs), fusion inhibitors (FIs).
In one embodiment, the antiretroviral (ART) agent is selected from the group
consisting
of Nucleoside reverse transcriptase inhibitors (NRTIs), Non-nucleoside reverse
transcriptase inhibitors (NNRTIs), Protease inhibitors (PIs), Integrase
inhibitors
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(INSTIs), Fusion inhibitors (FIs), Chemokine receptor antagonists (CCR5
antagonists)
and Entry inhibitors (CD4-directed post-attachment inhibitors).
Non-limiting examples of antiretroviral (ART) agents include:
- Nucleoside reverse transcriptase inhibitors (NRTIs) such as
e.g.:
o Abacavir (Ziagen0)
o Didanosine (Videx0, Videx0 EC)
o Emtricitabine (Emtriva)
o Lamivudine (Epivir0)
o Stavudine (Zerit0)
o Tenofovir disoproxil fumarate DF (Viread0)
o Tenofovir alafenamide AF
o Zidovudine (Retrovir0)
- Non-nucleoside reverse transcriptase inhibitors (NNRTIs) such
as e.g.:
o Delavirdine (Rescriptor0)
o Efavirenz (Sustiva0)
o Etravirine (Intelenee0)
o Nevirapine (Viramunee, Viramune0 XR)
o Rilpivirine (Edurant0)
o Doravirine (Pifeltro10)
- Protease inhibitors (PIs) such as e.g.:
o Atazanavir (Reyataz0)
o Darunavir (Prezista0)
o Fosamprenavir (Lexiva0)
o Indinavir (Crixivan0)
o Lopinavir/ritonavir (Kaletra0)
o Nelfinavir (Viracept0)
o Ritonavir (Norvir0)
o Saquinavir (Invirase0)
o Tipranavir (Aptivus0)
- Integrase inhibitors (INSTIs) such as e.g.:
o Raltegravir (IsentressO, Isentress HD)
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o Dolutegravir (Tivicay0)
o Elvitegravir (Vitekta0)
- Chemokine receptor antagonist (CCR5 antagonist) such as e.g.
o Maraviroc (Selzentry0)
- Fusion inhibitor (Fl) such as e.g.
o Enfuvirtide (Fuzeon0)
- Entry inhibitor such as e.g.
o lbalizumab (Trogarzo0)
- and any combination thereof.
In one embodiment, the ART agent according to the invention comprises several
ART
agents, or is a combination of several ART agents. These several ART agents
are for
instance chosen among the ART agents listed hereabove.
Generally, initial treatment regimens usually include two NTRIs combined with
a third
active antiretroviral drug, which may be in the INSTI, NNRTI, or PI class.
They may
sometimes include a booster, which may be cobicistat (Tybost0) or ritonavir
(Norvir0).
In one embodiment, the ART agent according to the invention comprises a
combination
of at least two ART agents, preferably chosen among the ART agents listed
hereabove.
In one embodiment, the ART agent according to the invention comprises a
combination
of two, three, four, five or six ART agents, preferably chosen among the ART
agents
listed hereabove.
ART combination products are known in the art and some of them are approved as
complete daily regimens.
Non-limiting examples of antiretro viral (ART) agents which are combination
ARTs or
combined ARTs (cARTs) include the following ART combinations:
- Elvitegravir + cobicistat + emtricitabine + tenofovir DF (Stribild0)
- Elvitegravir + cobicistat + emtricitabine + tenofovir AF (Genvoya0)
- Darunavir + cobicistat + emtricitabine + tenofovir AF (Symtuza0)
- Rilpivirine + emtricitabine + tenofovir AF (Odefsey0)
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- Rilpivirine + emtricitabine + tenofovir DF (CompleraC))
- Bictegravir + emtricitabine + tenofovir AF (Biktarvy10)
- Dolutegravir + abacavir + lamivudine (Triumecp)
- Dolutegravir + rilpivirine (JulucaC))
- Dolutegravir + lamivudine (Dovato0)
- Efavirenz + emtricitabine + tenofovir DF (Atripla0)
- Efavirenz + lamivudine + tenofovir DF (Symfi0)
- Doravirine + lamivudine + tenofovir DF (Delstrigoe)
- Emtricitabine + tenofovir AF (Descovy0)
- Emtricitabine + tenofovir DF (Truvada0)
- Abacavir + lamivudine (Epzicom0)
- Lamivudine + tenofovir DF (CimduoC:))
- Abacavir + lamivudine + zidovudine (TrizivirC))
- Zidovudine + lamivudine (Combivir0)
- Atazanavir + cobicistat (EvotazC))
- Darunavir ethanolate + cobicistat (PrezcobixC)).
In some embodiments, the subject has already received at least one dose of at
least one
antiretroviral (ART) agent, or is under antiretroviral therapy or under
combined
antiretroviral therapy (cART) comprising at least one antiretroviral (ART)
agent, before
being administered the combination of the invention. Said at least one
antiretroviral
(ART) agent may be the same antiretroviral (ART) agent as the one comprised in
the
combination of the invention, or it may be different from the antiretroviral
(ART) agent
comprised in the combination of the invention.
In one embodiment, said at least one antiretroviral (ART) agent is the same
antiretroviral
(ART) agent as the one comprised in the combination of the invention. In this
case, when
being treated according to the method of the invention, the subject goes on
receiving the
at least one antiretroviral (ART) agent he/she has already been given before,
while being
further administered a type III interferon blocking agent, an interferon-alpha
(IFN-a)
blocking agent, optionally an interferon-beta (IFN-1:3) blocking agent, and
optionally a
latency-reversing agent (LRA).
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In some embodiments, the subject has already received at least one dose of a
combined
antiretroviral therapy (cART) before being administered the combination of the
invention, and said cART comprises Nucleoside reverse transcriptase inhibitors
(NRTIs),
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) and Protease
inhibitors (PIs).
One option for eradicating HIV-1 reservoirs is based on HIV-1 reactivation in
latently-
infected cells while maintaining antiretroviral therapy (ART) in order to
prevent
spreading of the infection by the neosynthesized virus. Several latency
reversing agents
(LRAs) with distinct mechanistic classes have been characterized to reactivate
HIV-1
viral gene expression. Some LRAs have shown their potential to reverse HIV-1
latency
in order to purge latent HTV-1 .
Non-limiting examples of latency reversing agent (LRA) include:
- PKC agonists, which for instance act on NF-KB activation, such as e.g.
Prostratin
Bryostatin-1 Ingenols: Ingenol-B, Ingenol 3,20-dibenzoate (Ingenol-db),
ingeno1-3-angelate (ingenol mebutate, PEP005);
- MAPK agonist, which for instance act on MAP Kinase activation, such as
e.g. Procyanidin trimer Cl;
- CCR5 antagonist, which for instance act on NF-KB activation, such as
e.g. Maraviroc;
- Tat vaccine, which for instance act on Activation of HIV-1 LTR, such as
e.g. Tat Oyi vaccine, Tat-R5M4 protein;
- SMAC mimetics, which for instance act on Induction of non-canonical NF-KB
pathways, such as e.g. SBI-0637142, Birinapant;
- Inducers of P-TEFb release, which for instance act on Release of P-TEFb,
such as
e.g. BETis: JQ1, I-BET, I-BET151, OTX015, UMB-136, MMQO, CPI-203,
RVX-208, PFI-1, BI-2536 and BI-6727; HMBA;
- Activators of Akt pathway, which for instance act on Upregulation of Ala
signaling pathway, such as e.g. Disulfiram;
- Benzotriazole derivatives, which for instance act on STAT5 activation,
such as
e.g. 1-hydroxybenzotriazol (HOBO;
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- Epigenetic modifiers, which for instance act on HDAC inhibition, such as
e.g. HDACis: TSA, trapoxin, SAHA. romidepsin, panobinostat, entinostat,
givinostat, valproic acid, MRK-1/11, AR-42, fimepinostat, chidamide;
- Epigenetic modifiers, which for instance act on Suv39H1, G9a, SMYD2, such
as
5 e.g. HMTis: chaetocin, EPZ-6438, GSK-343, DZNEP, BIX-01294, UNC-0638;
- Epigenetic modifiers, which for instance act on DNMT1, 3a, 3b, such as
e.g. DNMTis: 5-AzaC, 5-AzadC; and
- Immunomodulatory LRAs, such as e.g. TLR agonists: TLR2 (Pam3CSK4), TLR7
(GS-9620), TLR8, TLR9 (MGN 1703) agonists; IL-15 agonist (ALT-803);
10 Immune checkpoint inhibitors: anti-PD-1 (nivolumab, pembrolizumab),
anti-CTLA-4 (ipilimumab).
In some embodiments, the latency reversing agent (LRA) is selected from the
group
consisting of PKC agonists, MAPK agonists, CCR5 antagonists, Tat vaccines,
SMAC
mimetics, inducers of P-TEFb release, activators of Akt pathway, benzotriazole
15 derivatives, epigenetic modifiers and immunomodulatory LRAs.
In some embodiments, the latency reversing agent (LRA) is a Prostratin
Bryostatin-1
Ingenol, such as Ingenol-B, Ingenol 3,20-dibenzoate (Ingenol-db), ingeno1-3-
angelate
(ingenol mebutate, PEP005); Procyanidin trimer Cl; Maraviroc; Tat Oyi vaccine,
Tat-R5M4 protein; SBI-0637142, Birinapant; a BETi such as JQ1, I-BET, I-
BET151,
20 0TX015, UMB-136, MMQO, CPI-203, RVX-208, PFI-1, BI-2536 and BI-6727
HMBA;
Disulfiram; 1-hydroxybenzotriazol (HOBt); a HDACi such as TSA, trapoxin, SAHA,
romidepsin, panobinostat, entinostat, givinostat, valproic acid, MRK-1/11, AR-
42,
fimepinostat, chidamide; a HMTi such as chaetocin. EPZ-6438, GSK-343, DZNEP,
B1X-01294, UNC-0638; a DNMTi such as 5-AzaC, 5-AzadC; a TLR agonist such as a
25 TLR2 agonist (Pam3CSK4), a TLR7 agonist (GS-9620), a TLR8 agonist, a
TLR9 agonist
(MGN 1703) agonist; an IL-15 agonist (ALT-803); and/or an immune checkpoint
inhibitor such as anti-PD-1 (nivolumab, pembrolizumab), or anti-CTLA-4
(ipilimumab).
In one embodiment, the latency reversing agent according to the invention
comprises
several LRAs, or is a combination of several LRAs, which are for instance
chosen among
30 the LRAs listed hereabove.
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In one embodiment, the latency reversing agent according to the invention
comprises a
combination of at least two LRAs, preferably chosen among the LRAs listed
hereabove.
In one embodiment, the latency reversing agent according to the invention
comprises a
combination of two, three, four, five or six LRAs, preferably chosen among the
LRAs
listed hereabove.
In one embodiment, at least one agent comprised in the combination is
comprised in a
composition.
In one embodiment, one, two or three agents selected from:
i) a type TTT interferon blocking agent,
ii) an interferon-alpha (IFN-a) blocking agent,
iii) optionally, an interferon-beta (IFN-f3) blocking agent,
are comprised in a single composition.
In one embodiment, one or two agents selected from:
iv) an antiretroviral (ART) agent, and
v) optionally, a latency-reversing agent (LRA).
are comprised in a single composition.
In one embodiment, all agents comprised in the combination (i.e. agents i),
ii), iii), iv)
and v)) are comprised in a composition.
In some embodiments, said composition consists essentially of the at least one
agent of
the combination according to the invention.
As used herein, "consisting essentially of", with reference to a composition,
means that
the at least one agent is the only therapeutic agent or agent with a biologic
activity within
said composition.
In one embodiment, said composition is a pharmaceutical composition and
further
comprises at least one pharmaceutically acceptable excipient.
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As used herein, the term ''excipient" refers to any and all conventional
solvents,
dispersion media, fillers, solid carriers, aqueous solutions, coatings,
antibacterial and
antifungal agents, isotonic and absorption delaying agents and the like. In
general, the
nature of the excipient will depend on the particular mode of administration
being
employed. For instance, parenteral formulations usually comprise injectable
fluids that
include pharmaceutically and physiologically acceptable fluids such as water,
physiological saline, balanced salt solutions, aqueous dextrose, glycerol or
the like as a
vehicle. For solid compositions (such as powder, pill, tablet, or capsule
forms),
conventional non-toxic solid carriers can include, for example, pharmaceutical
grades of
mannitol, lactose, starch, or magnesium stearate. In addition to biologically
neutral
carriers, pharmaceutical compositions to be administered can contain minor
amounts of
non-toxic auxiliary substances, such as wetting or emulsifying agents,
preservatives, and
pH buffering agents and the like, for example sodium acetate or sorbitan
monolaurate.
For human administration, preparations should meet sterility, pyrogenicity,
general safety
and purity standards as required by regulatory offices, such as, for example,
FDA Office
or EMA. In one embodiment, the excipient is an adjuvant, a stabilizer, an
emulsifier, a
thickener, a preservative, an antibiotic, an organic or inorganic acid or its
salt, a sugar, an
alcohol, an antioxidant, a diluent, a solvent, a filler, a binder, a sorbent,
a buffering agent,
a chelating agent, a lubricant, a coloring agent, or any other component
By "pharmaceutically acceptable" is meant that the ingredients of a
pharmaceutical
composition are compatible with each other and not deleterious to the subject
to which it
is administered. Examples of pharmaceutically acceptable excipient include,
but are not
limited to, water, saline, phosphate buffered saline, dextrose, glycerol,
ethanol and the
like or combinations thereof.
Pharmaceutically acceptable excipients that may be used in the pharmaceutical
combination of the invention include, but are not limited to, ion exchangers,
alumina,
aluminum stearate, lecithin, serum proteins, such as human serum albumin,
buffer
substances such as phosphates, glycine, sorbic acid, potassium sorbate,
partial glyceride
mixtures of saturated vegetable fatty acids, water, salts or electrolytes,
such as protamine
sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium
chloride,
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zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,
cellulose-based
substances (for example sodium carboxymethylcellulose), polyethylene glycol,
polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers,
polyethylene
glycol and wool fat.
In one embodiment, said composition is a vaccine composition. In one
embodiment, said
vaccine composition further comprises at least one adjuvant.
Another object of the invention is a pharmaceutical composition comprising a
combination of:
i) a type TTI interferon blocking agent,
ii) an interferon-alpha (IFN-a) blocking agent,
iii) optionally, an interferon-beta (IFN-f3) blocking agent,
and at least one pharmaceutically acceptable excipient, for use in the
treatment of AIDS
in a subject in need thereof.
Another object of the invention is a pharmaceutical composition comprising a
combination of:
iv) an antiretroviral (ART) agent, and
v) optionally, a latency-reversing agent (LRA).
and at least one pharmaceutically acceptable excipient, for use in the
treatment of AIDS
in a subject in need thereof.
The different agents of the combination or kit-of-parts according to the
invention
(i.e. parts i), ii), iii), iv) and v) of the combination) are to be
administered either
simultaneously. separately or sequentially with respect to each other.
According to one embodiment, the agent i), ii), iii), iv) or v) of the
combination, the
combination, the kit-of-parts, the composition or the pharmaceutical
composition of the
invention is formulated for administration to the subject.
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The expression "combined preparation" or "combination" refers to any
preparation
comprising at least two components, such as e.g. parts i), ii), iii), iv)
and/or v) of the
combination of the invention. The different components of the combined
preparation, or
of the combination, may be used simultaneously, semi-simultaneously,
separately,
sequentially or spaced out over a period of time so as to obtain the maximum
efficacy of
the combination.
For instance, they may be administered concurrently, i.e. simultaneously in
time, or
sequentially, i.e. one component is administered after the other one(s).
After administration of the first component, the other component(s) can be
administered
substantially immediately thereafter or after an effective time period. The
effective time
period is the amount of time given for realization of maximum benefit from the
administration of the components.
As a result, for the purposes of the present invention, the combined
preparations or
combinations are not limited to those which are obtained by physical
association of the
constituents, but may also be in the form of separate products permitting a
separate
administration, which can be simultaneous or spaced out over a period of time.
Alternatively, the different components may be co-formulated.
In one embodiment, the agent i), ii), iii), iv) or v) of the combination, the
combination,
the kit-of-parts, the composition or the pharmaceutical composition of the
invention may
be administered orally, intragastrically, parenterally, topically, by
inhalation spray,
rectally, nasally, buccally, preputially, vaginally Or via an implanted
reservoir.
In one embodiment, the administration of each part of the combination of the
invention
(i.e. agent i), ii), iii), iv) or v) of the combination of the invention) can
be done by the
same route of administration or by a different route of administration.
In one embodiment, the administration of i) a type III interferon blocking
agent, ii) an
interferon-alpha (IFN-a) blocking agent, and iii) optionally, an interferon-
beta (IFN-I3)
blocking agent, is done by a route of administration, and the administration
of iv) an
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antiretroviral (ART) agent and v) optionally, a latency-reversing agent (LRA)
is done by
another route of administration.
In one embodiment, the agent i), ii), iii), iv) or v) of the combination, the
combination,
the kit-of-parts, the composition or the pharmaceutical composition of the
invention is in
5 an adapted form for an oral or an intragastric administration. Thus, in
one embodiment,
the agent 0, ii), iii), iv) or v) of the combination, the combination, the kit-
of-parts, the
composition or the pharmaceutical composition of the invention is to be
administered
orally or intragastrically to the subject, for example as a powder, a tablet,
a capsule, and
the like or as a tablet formulated for extended or sustained release or as an
oral solution.
10 For instance, the antirctroviral (ART) agent and, optionally, the
latency-reversing agent
(LRA), are to be administered orally or intragastrically to the subject, for
example as a
powder, a tablet, a capsule, and the like or as a tablet formulated for
extended or sustained
release or as an oral solution.
Examples of forms adapted for oral or intragastric administration include,
without being
15 limited to, liquid, paste or solid compositions, and more particularly
tablets, tablets
formulated for extended or sustained release, capsules, pills, dragees,
liquids, gels, syrups,
slurries, suspensions, and the like.
In one embodiment, the antiretroviral (ART) agent and, optionally, the latency-
reversing
agent (LRA) are in an adapted form for an oral or intragastric administration.
Thus, in
20 one embodiment, the antiretroviral (ART) agent and, optionally, the
latency-reversing
agent (LRA) are to be administered orally or intragastrically to the subject,
for example
as a capsule or as a tablet or as an oral solution.
In another embodiment, the type III interferon blocking agent, the interferon-
alpha (IFN-
a) blocking agent and, optionally, the interferon-beta (IFN-13) blocking
agent, are in an
25 adapted form for an oral or intragastric administration. Thus, in one
embodiment, the type
III interferon blocking agent, the interferon-alpha (lFN-a) blocking agent
and, optionally,
the interferon-beta (IFN-13) blocking agent, are to be administered orally or
intragastrically to the subject, for example as a capsule or as a tablet or as
an oral solution.
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In one embodiment, the agent i), ii), iii), iv) or v) of the combination, the
combination,
the kit-of-parts, the composition or the pharmaceutical composition of the
invention is in
a form adapted for parenteral administration.
In another embodiment, the agent i), ii), iii), iv) or v) of the combination,
the combination,
the kit-of-parts, the composition or the pharmaceutical composition of the
invention is in
an adapted form for an injection such as, for example, for intravenous,
subcutaneous,
intramuscular, intraperitoneal intradermal, transdermal injection or infusion.
Thus, the
agent i), ii), iii), iv) or v) of the combination, the combination, the kit-of-
parts, the
composition or the pharmaceutical composition of the invention is to be
injected to the
subject, by intravenous, intramuscular, intraperitoneal, intrapleural,
subcutaneous,
transdermal injection or infusion.
A sterile injectable form may be a solution or an aqueous or oleaginous
suspension. These
suspensions may be formulated according to techniques known in the art using
suitable
dispersing or wetting agents and suspending agents. The sterile injectable
preparation
may also be a sterile injectable solution or suspension in a non-toxic
phaimaceutically
acceptable diluent or solvent. Among the acceptable vehicles and solvents that
may be
employed are water, Ringer's solution and isotonic sodium chloride solution.
In addition,
sterile, fixed oils are conventionally employed as a solvent or suspending
medium. For
this purpose, any bland fixed oil may be employed including synthetic mono- or
diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives
are useful in the
preparation of injectables, as are natural pharmaceutically acceptable oils,
such as olive
oil or castor oil, especially in their polyoxyethylated versions. These oil
solutions or
suspensions may also contain a long-chain alcohol diluent or dispersant, such
as
carboxymethyl cellulose or similar dispersing agents that are commonly used in
the
formulation of pharmaceutically acceptable dosage forms including emulsions
and
suspensions. Other commonly used surfactants, such as Tweens, Spans and other
emulsifying agents or bioavailability enhancers which are commonly used in the
manufacture of pharmaceutically acceptable solid, liquid, or other dosage
forms may also
be used for the purposes of formulation.
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In one embodiment, the type III interferon blocking agent, the interferon-
alpha (IFN-a)
blocking agent and, optionally, the interferon-beta (IFN-I3) blocking agent,
are in an
adapted form for a parenteral administration and/or injection. Thus, in
another
embodiment, the type III interferon blocking agent, the interferon-alpha (IFN-
cm) blocking
agent and, optionally, the interferon-beta (IFN-I3) blocking agent, are to be
administered
parenterally and/or injected to the subject, by intravenous, intramuscular,
intraperitoneal,
intrapleural, subcutaneous, transdermal injection or infusion, preferably by
intravenous
injection.
In another embodiment, the antiretroviral (ART) agent and, optionally, the
latency-reversing agent (LRA) are in an adapted form for a parenteral
administration
and/or injection. Thus, in another embodiment, the antiretroviral (ART) agent
and,
optionally, the latency-reversing agent (LRA) are to be administered
parenterally and/or
injected to the subject, by intravenous, intramuscular, intraperitoneal,
intrapleural,
subcutaneous, transdermal injection or infusion, preferably by intravenous
injection.
Preferably, the type III interferon blocking agent, the interferon-alpha (IFN-
a) blocking
agent and, optionally, the interferon-beta (IFN-I3) blocking agent, are in an
adapted form
for a parenteral administration and/or injection, and are to be administered
parenterally
and/or injected to the subject, by intravenous, intramuscular,
intraperitoneal, intrapleural,
subcutaneous, transdermal injection or infusion, preferably by intravenous
injection.
Preferably, the antiretroviral (ART) agent and, optionally, the latency-
reversing agent
(LRA) are in an adapted form for an oral or intragastric administration, and
are to be
administered orally or intragastrically to the subject, for example as a
capsule, a tablet or
an oral solution.
The different agents of the combination or kit-of-parts according to the
invention
(i.e. parts i), ii), iii), iv) and v) of the combination) are to be
administered either
simultaneously, separately or sequentially with respect to each other.
Parts i), ii), iii), iv) and v) of the combination may be administered
concurrently,
i.e. simultaneously in time, or sequentially, i.e. administration of certain
components of
the combination followed by administration of other components of the
combination.
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After administration of the first component(s), the other component(s) can be
administered substantially immediately thereafter or after an effective time
period.
The effective time period is the amount of time given for realization of
maximum benefit
from the administration of the components.
In one embodiment, the type III interferon blocking agent, the interferon-
alpha (IFN-a)
blocking agent, optionally the interferon-beta (IFN-13) blocking agent, the
antiretroviral
(ART) agent and, optionally, the latency-reversing agent (LRA) are all
administered at
the same time.
In one embodiment, the type III interferon blocking agent, the IFN-a blocking
agent, and
optionally the interferon-beta (IFN-f3) blocking agent arc administered
concurrently or
simultaneously.
In one embodiment, the antiretroviral (ART) agent, and optionally the latency-
reversing
agent are administered concurrently or simultaneously.
In one embodiment, the type III interferon blocking agent, the interferon-
alpha (IFN-a)
blocking agent, and/or the interferon-beta (IFN-13) blocking agent, are to be
administered
prior to the antiretroviral (ART) agent and/or the latency-reversing agent
(LRA).
In one embodiment, the antiretroviral (ART) agent and/or the latency-reversing
agent
(LRA) are to be administered prior to the type ITT interferon blocking agent,
the interferon-
alpha (IFN-a) blocking agent, and/or the interferon-beta (IFN-13) blocking
agent.
In one embodiment, the subject receives one or more doses (one or more takes)
of the
type ITT interferon blocking agent, the IFN-a blocking agent, and/or the
interferon-beta
(IFN-13) blocking agent before starting receiving all parts i), ii), iii), iv)
and v) of the
combination.
For instance, in a first period of time, the subject receives one or more
doses (one or more
takes) of the type TTT interferon blocking agent, the IFN-a blocking agent,
and optionally
the interferon-beta (IFN-13) blocking agent, during one or several weeks (e.g.
1, 2, 3, 4, 5,
6, 7, 8 or 10 weeks) in the absence of ART agent and LRA administration. Then,
in a
second period of time, the subject continues receiving administrations of the
type ITT
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interferon blocking agent, the IFN-a blocking agent, and/or the interferon-
beta (IFN-(3)
blocking agent, and also receives administrations of the antiretroviral (ART)
agent, and
optionally the latency-reversing agent.
Alternatively, in a first period of time, the subject receives one or more
doses (one or
more takes) of the antiretroviral (ART) agent, and optionally the latency-
reversing agent,
during one or several weeks or months (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 10 or 12
weeks or months)
in the absence of administration of the type III interferon blocking agent,
the IFN-a
blocking agent, and/or the interferon-beta (IFN-I3) blocking agent. Then, in a
second
period of time, the subject continues receiving administrations of the
antiretroviral (ART)
agent, and optionally the latency-reversing agent, and also receives
administrations of the
type III interferon blocking agent, the IFN-a blocking agent, and optionally
the
interferon-beta (IFN-I3) blocking agent.
In one embodiment, the agent i), ii), iii), iv) or v) of the combination, the
combination,
the kit-of-parts, the composition or the pharmaceutical composition of the
invention is to
be administered to the subject in need thereof in a therapeutically effective
amount.
The term "therapeutically effective amount", as used herein, refers to an
amount
effective, at dosages and for periods of time necessary, to achieve a desired
preventive
and/or therapeutic result.
It will be however understood that the total daily usage of the agent i), ii),
iii), iv) or v) of
the combination, the combination, the kit-of-parts, the composition or the
pharmaceutical
composition of the invention will be decided by the attending physician within
the scope
of sound medical judgment. The specific therapeutically effective dose level
for any
particular patient will depend upon a variety of factors including the disease
being treated
and the severity of the disease; activity of the specific agent(s), the
combination, the kit-
of-parts, the pharmaceutical composition or medicament employed; the age, body
weight,
general health, sex and diet of the subject; the time of administration, route
of
administration, and rate of excretion of the specific agent(s), the
combination, the
kit-of-parts, the pharmaceutical composition or medicament employed; the
duration of
the treatment; drugs used in combination or coincidental with the specific
agent(s), the
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combination, the kit-of-parts, the pharmaceutical composition or medicament
employed;
and like factors well known in the medical arts. For example, it is well
within the skill of
the art to start doses of the compound at levels lower than those required to
achieve the
desired therapeutic effect and to gradually increase the dosage until the
desired effect is
5 achieved. The total dose required for each treatment may be administered
by multiple
doses or in a single dose.
In one embodiment, a therapeutically effective amount of one of the agents i),
ii), iii), iv)
or v) of the combination of the invention ranges from about 0.1 mg/kg to about
10 mg/kg,
from about 0.2 mg/kg to about 9 mg/kg, from about 0.3 mg/kg to about 8 mg/kg,
from
10 about OA mg/kg to about 7.5 mg/kg, from about 0.5 mg/kg to about 7
mg/kg.
In one embodiment, a therapeutically effective amount of the type III
interferon blocking
agent, the interferon-alpha (IFN-cm) blocking agent, or the interferon-beta
(IFN-I3)
blocking agent (i.e. of one of the agents i), ii), iii)) ranges from about 1
mg/kg to about
10 mg/kg, from about 2 mg/kg to about 9 mg/kg, from about 3 mg/kg to about 8
mg/kg,
15 from about 4 mg/kg to about 7 mg/kg, from about 4 mg/kg to about 6
mg/kg.
In one embodiment, a therapeutically effective amount of one of the agent i),
ii), iii), iv)
or v) of the combination of the invention ranges from about 1 mg to about 4000
mg, from
about 10 mg to about 3000 mg, from about 25 mg to about 2000 mg, from about 50
mg
to about 2000 mg, from about 100 mg to about 1500 mg, from about 200 mg to
about
20 1000 mg.
In one embodiment, a therapeutically effective amount of the antiretroviral
(ART) agent
or of the latency-reversing agent (LRA) (i.e. of one of the agents iv) or v))
ranges from
about 1 mg to about 4000 mg, from about 10 mg to about 3000 mg, from about 25
mg to
about 2000 mg, from about 50 mg to about 2000 mg, from about 100 mg to about
25 1500 mg, from about 200 mg to about 1000 mg.
Usually, there is a set time interval between separate administrations of the
agent i), ii),
iii), iv) or v) of the combination, the combination, the kit-of-parts, the
composition or the
pharmaceutical composition of the invention. While this interval varies for
every subject,
typically it ranges from 1 days to several weeks, and is often 1, 2, 4, 6 or 8
days, or 1, 2,
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4, 6 or 8 weeks. In one embodiment, the administration regimes typically have
from 1 to
20 administrations of the different parts of the combination of the invention,
but may have
as few as one or two or four or eight or ten. In another embodiment, the
administration
regime is annual, biannual or other long interval (5-10 years).
In one embodiment, a therapeutically effective amount of the agent i), ii),
iii), iv) or v) of
the combination, the combination, the kit-of-parts, the composition or the
pharmaceutical
composition of the invention is to be administered once a day, twice a day,
three times a
day or more.
In one embodiment, a therapeutically effective amount of the agent i), ii),
iii), iv) or v) of
the combination, the combination, the kit-of-parts, the composition or the
pharmaceutical
composition of the invention is to be administered every day, every two days,
every three
days, every four days, every five days, every six days.
In one embodiment, a therapeutically effective amount of the agent i), ii),
iii), iv) or v) of
the combination, the combination, the kit-of-parts, the composition or the
pharmaceutical
composition of the invention is to be administered every week, every two
weeks, every
three weeks, every four weeks, every five weeks.
In one embodiment, a therapeutically effective amount of the agent i), ii),
iii), iv) or v) of
the combination, the combination, the kit-of-parts, the composition or the
pharmaceutical
composition of the invention is to be administered every month, every two
months, every
three months, every four months, every five months, every six months.
In one embodiment, a therapeutically effective amount of the agent i), ii),
iii), iv) or v) of
the combination, the combination, the kit-of-parts, the composition or the
pharmaceutical
composition of the invention is to be administered every 12 hours, every 24
hours, every
36 hours, every 48 hours, every 60 hours, every 72 hours, every 96 hours.
In a preferred embodiment, a therapeutically effective amount of the type III
interferon
blocking agent, the interferon-alpha (IFN-a) blocking agent, or the interferon-
beta (IFN-
p) blocking agent (i.e. of one of the agents i), ii), iii)) is to be
administered every week,
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every two weeks, every three weeks, every four weeks, every five weeks,
preferably every
two weeks.
In a preferred embodiment, a therapeutically effective amount of the
antiretroviral (ART)
agent or of the latency-reversing agent (LRA) (i.e. of one of the agents iv)
or v)) is to be
administered daily, for instance once a day, twice a day, or three times a
day.
In a preferred embodiment, a therapeutically effective amount of the
antiretroviral (ART)
agent or of the latency-reversing agent (LRA) (i.e. of one of the agents iv)
or v)) is a daily
dose to be administered in one, two, three or more takes or in one, two, three
or more
injections.
In a preferred embodiment, the antiretroviral (ART) agent and optionally the
latency-
reversing agent are administered daily, and the IFN-a. blocking agent, the
type Ill
interferon blocking agent, and optionally the interferon-beta (IFN-f3)
blocking agent, are
administered every three weeks.
In one embodiment, the agent i), ii), iii), iv) or v) of the combination, the
combination,
the kit-of-parts, the composition or the pharmaceutical composition of the
invention is for
acute administration. Preferably, the agent i), ii), iii), iv) or v) of the
combination, the
combination, the kit-of-parts, the composition or the pharmaceutical
composition of the
invention is for chronic administration.
In one embodiment, a therapeutically effective amount of the agent i), ii),
iii), iv) or v) of
the combination, the combination, the kit-of-parts, the composition or the
pharmaceutical
composition of the invention is to be administered for a period of time
ranging from about
two weeks to about twenty-four weeks, from about two weeks to about twelve
weeks,
from about two weeks to about six weeks.
In one embodiment, a therapeutically effective amount of the agent i), ii),
iii), iv) or v) of
the combination, the combination, the kit-of-parts, the composition or the
pharmaceutical
composition of the invention is to be administered for about 1 month, 2
months, 3 months,
6 months, 1 year or more.
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Alternatively, a therapeutically effective amount of the agent i), ii), iii),
iv) or v) of the
combination, the combination, the kit-of-parts, the composition or the
pharmaceutical
composition of the invention is to be administered to the subject until no
cell containing
replication-competent proviral HIV DNA is detected in a blood sample from the
subject.
Indeed, in one embodiment, the method of treatment of the invention further
comprises
assessing the presence of cells containing replication-competent proviral HIV
DNA in a
blood sample from the subject.
In another embodiment, the presence of cells containing replication-competent
proviral
HIV DNA is assessed in a blood sample from the subject.
In some embodiments, a therapeutically effective amount of the agent i), ii),
iii), iv) or v)
of the combination, the combination, the kit-of-parts, the composition or the
pharmaceutical composition of the invention is to be administered to the
subject until no
cell containing replication-competent proviral HIV DNA is detected in a blood
sample
from the subject.
Cells containing replication-competent proviral HIV DNA, also called
"resistant cells"
or "reservoir cells" may be detected in a sample, or their quantity, level or
frequency in
a sample may be measured by various assays known by the person skilled in the
art.
For instance, cells containing replication-competent proviral HIV DNA may be
detected
in a sample, or their quantity, level or frequency in a sample may be measured
by ex vivo
viral outgrowth assay.
For example, plasma 1-ITV-1 RNA levels, size of the HIV reservoir and/or CD4+
T cell
count may be typically monitored for instance every 2 weeks, after collection
of blood
samples, 1 day before administration of the interferon-blocking agents (i.e.
parts i), ii)
and iii) of the combination described herein).
Plasma HIV-1 RNA levels may for instance be determined using the Roche COBAS
AmpliPrep/COBAS TaqMan 1-IIV-1 Assay (version 2.0) or the Roche cobas HIV-1
quantitative nucleic acid test (cobas 6800), which quantitate HIV-1 RNA over a
range of
2x101 to 1x107 copies/ml.
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Size of the HIV reservoir may for instance be assessed with Quantitative Viral
Outgrowth
Assay (QVOA).
QVOA may typically be performed as previously described in Huang SH et al,
2018 J Clin Invest 128:876-889. Briefly, isolated CD4+ T cells may typically
be plated
out in serial dilutions (e.g. 2, 1, 0.5, 0.2, and 0.1 million per well), for
instance into
12 wells in 24-well plates with added phytohemagglutinin (PHA; e.g. 2 kg/m1)
and
irradiated HIV-negative donor PBMC (e.g. 2 x 106 cells/well) to reactivate the
infected
cells. MOLT-4 CCR5 cells (e.g. 2 x 106 cells/well) may typically be added
after 2 days
of culture, to amplify the HIV. The p24 antigen in the culture supernatant may
typically
be quantified after 2 weeks of culture, for instance using an HIV p24 antigen
enzyme-linked immunosorbent assay (ELISA) kit (Perkin-Elmer, Hopkinton, MA).
Estimated frequencies of cells with replication-competent HIV-I may typically
be
calculated using limiting dilution analysis.
CD4+ T-cell counts may for instance be determined by a clinical flow cytometry
assay.
As a non-limiting example, administration of the interferon-blocking agents
(i.e. parts i),
ii) and iii) of the combination described herein) may be stopped when no cells
with
replication-competent HIV-1 are detected following 2-3 consecutive limiting
dilution
virus outgrowth assays (VOA).
As a non-limiting example, administration of the antiretroviral (ART) agent
or/and the
latency-reversing agent (LRA) (i.e. parts iv) and/or v) of the combination
described
herein) may be stopped when no cells with replication-competent HIV-1 are
detected with
3-4 consecutive limiting dilution virus outgrowth assays (VOA).
It will be apparent to the person skilled in the art that such timing in the
treatment
interruption is flexible.
In some cases, if viral clearance was not complete, AIDS symptoms (or a
detectable viral
load) may appear anew after a certain time after interruption of the
treatment, such as
e.g. after a few months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years,
etc.
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after interruption of the treatment. In these cases, treatment with the
combination of the
invention may then be resumed after a certain time of treatment interruption.
Thus, in one embodiment, the combination of the invention is administered to
the subject
for a first period of time, then said treatment is interrupted during a period
of time of a
5 few months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 8 years
or 10 years, and
then the combination of the invention is administered anew to the subject for
a second
period of time, for instance until no cell containing replication-competent
proviral HIV
DNA is detected in a blood sample from the subject.
The subject may thus alternate between periods of time during which he
receives
10 administrations of the combination described herein and periods of time
of treatment
interruption.
The combination, the kit-of-parts, the composition or the pharmaceutical
composition as
described herein may be used alone.
Thus, in one embodiment, the combination, the kit-of-parts, the composition or
the
15 pharmaceutical composition as described herein is used alone and
comprises a
therapeutically effective dose of each part each part of the combination (i.e.
agent i), ii),
iii), iv) or v)).
In another embodiment, the combination, the kit-of-parts, the composition or
the
pharmaceutical composition as described herein is used in combination with at
least one
20 further therapeutic agent.
Such administration may be simultaneous, separate or sequential. For
simultaneous
administration the agents may be administered as one composition or as
separate
compositions, as appropriate. The further therapeutic agent is typically
relevant for
disorders to be treated.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Antiviral activity of type I and type III interferons. (A)
Expression of ISGs in
HepG2. HepG2 cells were treated with IFNa2a or IFN2J-4 (10 ng/ml). After 4 h
of
stimulation, qRT-PCR were used to examine the mRNA levels of the interferon-
induced
genes, IFIT1, MX1 and OASL and fold-changes was calculated by 2-AA1t method as
compared with non-treated cell control and using endogenous S14 mRNA level for
normalization. (B) Antiviral activity of type I and III IFNs against EMCV.
IFNa2a or
IFN21/2/3/4 (10 ng/ml) were added to HepG2 cells 24 h prior to challenge with
EMCV.
Forty-eight after infection with EMCV, cells were assayed for viability with a
bioassay.
A570 values were directly proportional to cell viability and therefore
antiviral activity of
the respective IFNs. IFN-a treatment without viral challenge was used as a
baseline of
the viability of the cells.
Figure 2. Anti-proliferative activity of type I and type III interferons
against CD4+
T cells. CFSE-stained CD4+ T cells (10x104/well) were stimulated for 5 days in
96 round-
bottomed microwells with allogeneic poly I:C matured DC in absence (control)
or
presence of 10 ng/ml of IFN-a2a, or IFNX1 or IFN22 or IFN23 or IFN2.4. When
indicated,
anti-interferon type I receptor antibody was added. The percentage of CFSE
dilution was
evaluated by flow cytometry.
Figure 3. IFN-a2a but not IFN-type III induces the expression of IS Gs in CD4+
T cells.
CD4+ T cells were treated with IFNa2a or IFNX1/2/3/4 (10 ng/ml). After 4 h of
stimulation, qRT-PCR were used to examine the mRNA levels of the interferon-
induced
genes, IFIT1, MX1 and OASL and fold-changes was calculated by 2-AAct method as
compared with non-treated cell control and using endogenous S14 mRNA level for
normalization.
Figure 4. IFN-a2a but not IFN-type III stimulates the phosphorylation of Statl
in CD4+
T cells. CD4+ T cells were stimulated with 10 ng m1-1 of IFN-2d, IFN-22, IFN-
23,
IFN-2.,4, or IFN-a2a for 20 min, or were left unstimulated (control).
Increases in pSTAT1
were evaluated as a ratio of induction over baseline levels
(MFI fold change = MFI cytokine-stimulated/MFI untreated cells)
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Figure 5. IFN-a2a but not IFN-type III increases CD38 expression in CD3/CD28
stimulated CD4+ T cells. CFSE-stained CD4+ T cells (4x104/well) were cultured
in
96 round-bottomed microwells in the presence of ACD3-feeder (4x104/well) and
plate-bound anti-CD3 mAb (2 g/ml), soluble anti-CD28 mAb (2 ug/m1) with
increasing
dose of IFN-a2a or IFN type III. CD38 Median Fluorescence Intensity (MFI) was
measured by flow cytometry in CD3+ 7-AAD-CFSE+ stimulated CD4+ T cells at the
end
of the culture.
Figure 6. Comparison of CM CD8+ T cell distributions and senun IFN-ct levels
in HIV-
1-infected subjects and critical pathogenic role of IFN-a in human HIV-1
infection.
(A) Comparison of CM CD8+ T cell distributions in HIV-1-infected subjects;
(B) Comparison of serum IFN-ct levels in HIV-1-infected subjects; (C)
Relationship
between CD8+ CM frequency and serum IFN-a level in non-treated HIV patients
(EC and pre-cART group).
Figure 7. Serum levels of IFN-a and IFN-X, before and after cART treatment in
HIV-1
patients originating from two distinct institutes ((A) Institute 1; (B)
Institute 2) and
control healthy donors.
Figure 8. Schematic diagram of possible administration schedule for the
combination of
the invention.
Figure 9: HIV infection of NCG humanized mice induces 1FN-1 and 1FN 111
production.
NCG humanized mice were intraperitoneally injected with HIV-NL4.3. Blood and
spleen
were removed from infected and non-infected humanized NCG mice at day 2 post-
infection, when the virus load has reached a plateau at day 10 and at day 30.
Graphs show
1FN-a, IFN-X3 and IFN-X1 mRNA levels in isolated PBMCS (A, B, C) and
splenocytes
(D, E, F) respectively.
Figure 10: Activated CD4+ T cells express IFN-lambda Receptor. CD4+ T cells
were
cultured in presence of IL-2 without (black bars) or with anti-CD3 and anti-
CD28
antibodies (1 pg/ml each) (grey bars) for 3 days. IFNLR1 (IL28RA) and ILlORB
expression was determined by RT-qPCR (A, B) and the detection of fixation of
IFN-X3
on cultured CD4+ T cells (C) was assessed by IFN-X3 MFI measured by Flow
cytometry.
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1FIH1 (D) and IF127 (E) mRNA induction in IFN-X3-treated CD4+ T cells cultured
as
described above was determined by RT-qPCR.
Figure 11: IFN-k3 inhibits HIV- l infection of activated CD4+ T cells. 3 day-
CD3/CD28/IL2-stimulated CD4+ T cells were treated with the indicated reagents
IFN-
X3, IFN-sa, anti-IFNLR1 (IL28RA), anti-IFNAR2 neutralizing antibodies. 24
hours after,
treated cells were infected with HIV NL4.3. Graphs show the frequency of
infected cells
5 days post-infection using intracellular Gag p24 staining (n = 4 donors).
Figure 12: Sequential treatment with two latency-reversing agents, DNA
methylation
inhibitor 5-AzadC and HDACI MS-275, induce IFN-1 Receptor expression on CD4+ T
cells. 3-day-CD3/CD28/IL2-stimulated CD4+ T cells and J-Lat 10.6 cells were
mock-
treated or treated with 5- AzadC for 72 h with MS-275 for the last 24 h. At 72
h post-
treatment, mock (black bars) and 5-AzadC/MS-275 (grey bars) CD4+ T (A, B, C,
D) and
J-Lat 10.6 cells (E, F, G, H) were harvested and first analyzed for their
capacity to express
1FNLR1 (IL28RA) by RT-qPCR (A and E). Then samples were cultured with IFN-23.
The presence of IFN-X3 binding on T cells was assessed by IFN-X3 MFI measured
by
Flow cytometry (B and F). The T cells' sensitivity to IFN- X3 was evaluated by
the
identification of IFN-stimulated gene (ISG) proteins IFIHI (C and G) and IF127
(D and
H) by RT-qPCR.
Figure 13: The NtRTI TDF enhances the production of IFN-X3 by HT-29 cells.
Colon
cancer HT-29 cells were incubated with dimethyl sulfoxide (DMSO) or tenofovir
disoproxil fumarate (TDF, 25 ItM). After 48 hours, IFN-X3 levels in treated HT-
29 cells
were evaluated by ELISA.
Figure 14: Experimental scheme for assessing inhibition of HIV infection in
stimulated
CD4+ T cells by IFN-X3 released by TDF treated HT-29 cells. HT-29 cells were
cultured
in the bottom chamber of Transwell plates. When the culture was subcontinent,
Tenofovir
disoproxil fumarate (TDF) was added. After 24 hours of stimulation. activated
CD4 T
cells were deposited in the insert which is then placed in the culture well so
as to be
immersed in the culture medium of the HT-29 cells. When indicated, the CD4+ T
cells
were pre-treated with an IFNLR1 (IL28RA) neutralizing antibody. After 24 hours
of co-
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culture, the cells in the insert were harvested, infected and then placed back
into the co-
culture for an additional 5 days of culture. The frequency of infected cells
was determined
by intracellular HIV p24 by FACS.
Figure 15: IFN-2c3 secreted by the NtRTI TDF-treated HT-29 cells impairs CD4+
T cell
infection. Experiments were performed using 12-transwell chambers with a
polycarbonate filter (0.2 na pore size). HT-29 were grown until subconfluence
in the
lower chamber. Then HT-29 cells were treated with tenofovir disoproxil
fumarate (TDF).
After 24h of TDF treatment, 3-day-CD3/CD28/IL2- stimulated CD4+ T cells were
seeded
in the upper chamber in presence of IL-2. When indicated, anti-IFNLR1 (IL28RA)
neutralizing Ab was added to the culture. After 24h of co-culture, CD4+ T
cells were
isolated, infected with HIV NL4.3 and added again to the upper chamber. Graphs
show
the frequency of infected cells 5 days post-infection using intracellular Gag
p24 staining
(n = 3 donors).
Figure 16: Experimental scheme for assessing the inhibition of HIV latency
reversal in
latently infected J-Lat cells by IFN-X3 released by TDF treated HT-29 cells.
HT-29 cells
were cultured in the bottom chamber of Transwell plates. When the culture was
subconfluent. Tenofovir disoproxil fumarate (TDF) was added. After 24 hours of
stimulation, J-Lat 10.6 cells pretreated with 5-AzadC and MS-275 are deposited
in the
insert which was then placed in the culture well. When indicated, the J-Lat
cells were pre-
incubated with an IFNLR1 (IL28RA) neutralizing antibody. After 48 hours of co-
culture,
HIV reactivation was monitored as the percentage of living GFP-positive cells
by Flow
Cytometry analysis.
Figure 17: IFN-X3 released by TDF-treated HT-29 cells inhibits in vitro HIV
reactivation
in latently infected J-Lat clone 10.6. Experiments were performed using 12-
transwell
chambers with a polycarbonate filter (0.2 p.m pore size). HT-29 were grown
until
subconfluence in the lower chamber. Then HT-29 cells were treated with
tenofovir
disoproxil fumarate (TDF). After 24 h of TDF treatment, J-Lat clone 10.6 cells
which had
been pretreated for 72 hours with 5-AzadC and MS-275 as described above were
seeded
in the upper chamber. When indicated, anti-IFNLR1 (IL28RA) Ab was added to the
culture. After 48 hours of co-culture, HIV reactivation was measured by
quantification of
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GFP expression by flow cytometry (n = 4 donors).
Figure 18. Schematic diagram of pre-clinical protocol for HIV-1 reservoirs
elimination
in HIV-1 infected humanized mice.
5 EXAMPLES
The present invention is further illustrated by the following examples.
Example 1: Effects of type I and type III interferons on innate and adaptative
immune responses
Material and Methods
10 Human cell lines
HCC HepG2 and normal kidney epithelial Vero cell lines were obtained from
ATCC.
Cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10%
heat-inactivated Fetal Bovine Serum, 2 mM L-glutamine, 1% penicillin and
streptomycin
solution in hypoxia 2%. Cancer cell lines were grown to 70-100% confluency,
15 subsequently passaged for a maximum of 5 times and freshly
thawed thereafter.
Cells were detached by means of accutase, resuspended in FBS-containing medium
and
collected by means of centrifugation (300g, 3min). Cell numbers were
determined by
means of trypan blue.
Human Blood Sample
20 Blood samples from healthy individuals originated from
Etablissement Francais du Sang
(EFS, Paris). Blood cells are collected using standard procedures.
Cell Purification and culture
Peripheral blood mononuclear cells (PBMCs) are isolated by density gradient
centrifugation on Ficoll-Hypaque (Pharmacia). PBMCs are used either as fresh
cells or
25 stored frozen in liquid nitrogen. T-cell subsets and T cell-
depleted accessory cells (ACD3
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cells) are isolated from either fresh or frozen PBMCs. T cell¨depleted
accessory cells
(ACD3 cells) are isolated by negative selection from PBMCs by incubation with
anti-
CD3¨coated Dynabeads (Dynal Biotech) and are irradiated at 3000 rad (referred
to as
ACD3-feeder). CD4+ T cells are negatively selected from PBMCs with a CD4+ T-
cell
isolation kit (Miltenyi Biotec), yielding CD4+ T-cell populations at a purity
of 96-99%.
T cell subsets are cultured either in IMDM supplemented with 5% SVF,
100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential
amino
acids, glutamax and 10 mM HEPES (IMDM-5 media) in hypoxia 2%.
Freezing and thawing of cells
Cells were frozen in FBS containing 10% DMSO. Cryotubes were placed in
CoolCell
(Biocision) freezing containers and incubated at -80 C. After 2 days tubes
were
transferred to liquid nitrogen and stored until required. Thawing of cells was
performed
by placing cryotubes in a 37 C water bath for approximately 30 seconds. Next,
cell
suspension was mixed with equivalent volume of pre-warmed media and
subsequently
transferred to falcon tubes containing the same medium. Cells were pelleted by
centrifugation (300g, 3min) to remove DMSO. The cell pellet was resuspended in
cell
culture medium
Real-time PCR for ISas. detection
HepG2 cells were seeded at a density of 2 x 105 cells per well in 12-well
plates and
incubated for 24 h. Then, fresh media was added with the indicated
interferons. The cells
were incubated for 4 h and then lysed, and RNA was purified using an
extraction kit
(Qiagen), according to the manufacturer's instructions. Synthesis of cDNA was
performed using the PrimeScript RT Reagent kit (TAKARA). Quantitative PCR was
carried out using the Power SYBR Green PCR Master Mix (Applied Biosystems) on
a
LightCycler 480 instrument (Roche). Each reaction was carried out in duplicate
in a total
volume of 100 [IL. Primers were designed to be intron-spanning using Primer3
or Primer
Express v3.0 software (Applied Biosystems). To measure the cellular
transcriptional
response to IFN stimulation, 3 ISG targets, MXI, OASL and ISG15, were selected
based
on published results investigating the transcriptional response in IFN-
stimulated PBMCs
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(see, for example, Waddell et al. (2010) PLoS One. 5(3):e97532). For gene
induction
assays, fold change values were calculated using the AACt method. The
geometric mean
of the Ct values of the reference genes, S14, was used as a reference value.
Virus production
The virus used EMCV (FA strain) was grown on monolayers of Vero cells to
complete
cytopathic effect or until all cells were affected by the infection as
determined by
microscopy and prepared by two cycles of freezing and thawing, followed by
centrifugation for 30 min at 5,000 x g for removal of cellular debris.
Antiviral assay
Antiviral assays were done on HepG2 cells, which were seeded in DMEM
supplemented
with 10% FCS at a density of 1.5 x 104 in 96-well plates and left to settle.
The cells were
incubated with indicated doses of IFNs for 24 h before challenge with EMCV.
The cells
were incubated with virus for 48 h. The medium was removed between each step.
The
viability of the cells was analyzed by a bioassay based on the dehydrogenase
system; this
system in intact cells will convert the substrate, MTT, into formazan (blue),
which in turn
can be measured spectrophotometrically. Briefly, the cells were given MTT and
incubated for 2 h. An extraction buffer (containing 6 to 11% sodium dodecyl
sulfate and
45% N, N-dimethylformamide) was added to the cells, and the cells were then
incubated
overnight at 37 C. Subsequently, the absorbance at 570 nm was determined
employing
the extraction buffer as the blank probe. A570 was directly proportional to
antiviral
activity.
Flow cytornetry analysis
CD3+ T cells staining: anti-CD4 (SK3)-APC, anti-CD3 (UCHT1)-FITC, anti-CD8
(RPA-T8)-BV421 are from Becton Dickinson. Cells are stained for surface
markers
(at 4 C in the dark for 30 min) using mixtures of Ab diluted in PBS containing
3% FBS,
2mM EDTA (FACS buffer).
STAT1 signaling analysis: Flow cytometry analysis of STAT1 phosphorylation
(pSTAT1) was conducted in CD4 T cells by using BD Phosflow technology
according
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to the manufacturer's instructions (BD Bio-sciences, San Jose, CA). CD4+ T
cells were
stimulated by incubation with interferon type I and Type III at 37 C for 20
min or left
untreated. Activation was stopped by fixation using BD Phosflow Lyse/Fix
Buffer
(BD Biosciences) and cells were permeabilized with BD Penn Buffer III
(BD Biosciences). Cells were stained with antibody recognizing specific
phosphorylated
STAT tyro sines: p-STAT1 (Y701)-PE. In multiparametric immunophenotyping
experiments, cells were simultaneously stained with anti-CD3-FITC and 7-AAD.
Increases in pSTAT1 were assayed as a ratio of induction over baseline levels
(MFI fold change = MFI cytokine-stimulated/MFI untreated cells)
CFSE staining: CD4+ T cells were stained with 1 pM CFSE (CellTrace cell
proliferation
kit; Molecular Probes/Invitrogen) in PBS for 8 min at 37 C at a concentration
of
1.107 cells/ml. The labeling reaction was stopped by washing twice the cell
with
RPMI-1640 culture medium containing 10% FBS. The cells were then re-suspended
at
the desired concentration and subsequently used for proliferation assays.
7-AAD staining: Apoptosis of stimulated CFSE-labeled CD4+ T was determined
using
the 7-AAD assay. Briefly, cultured cells were stained with 20 pg/mL nuclear
dye
7-amino-actinomycin D (7-AAD; Sigma-Aldrich, St-Quentin Fallavier, France) for
30 minutes at 4 C. FSC/7-AAD dot plots distinguish living (FSCing11/7-AAD-)
from
apoptotic (FSChighn-AAD+) cells and apoptotic bodies (FSC1 w/7-AAD+) and
debris
((FSCI0nv/7-AAD-). Living cells were identified as CD3+ 7-AAD-FSC+ cells.
Appropriate isotype control Abs are used for each staining combination.
Samples are
acquired on a BD LSR FORTESSA flow cytometer using BD FACSDIVA 8Ø1 software
(Becton Dickinson). Results are expressed in percentage (%) or in mean
fluorescence
intensity (MFI).
Functionnal assay
T cell proliferation: T cell proliferation was assessed with CFSE-dilution
assays.
For CFSE-dilution assay, at coculture completion, stimulated CFSE-labeled CD4+
T cells
were harvested, co-stained with anti-CD3 mAb and 7-AAD, and the percentage of
proliferating cells (defined as CFSE low fraction) in gated CD3+ 7-AAD- cells
was
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determined by flow cytometry.
T cell activation: CD38 Median Fluorescence Intensity (MFI) of CD38 expression
was
measured by flow cytometry in CD3+ 7-AAD-CFSE+ stimulated CD4+ T cells at the
end
of the culture.
CD4+ T cell polyclonal stimulation: CFSE-stained CD4+ T cells (5x104/well)
were
cultured in 96 round-bottomed microwells in the presence of ACD3-feeder
(1x105/well)
and plate-bound anti-CD3 Ab (2 pg/nal), soluble anti-CD28 mAb (2 pg/m1). CD4+
T cell
proliferation was evaluated with CFSE dilution assays as described above by
flow
cytometry. Cells were stimulated in presence of different amounts of
recombinant
cytokines.
Allogeneic mixed lymphocyte reaction: CFSE-stained CD4+ T cells (5x104/we11)
were
cultured in 96 round-bottomed microwells in the presence of allogeneic mature
DC.
Proliferation of allo-activated CD4+ T cells with CFSE dilution assays as
described above
by flow cytometry. Cells were stimulated in presence of different amounts of
recombinant
cytokines.
Statl phosphorylation analysis: CD4 T cells were stimulated with IFN-M,
IFN-24, or IFN-a2a (10 ng/ml) for 20 min, or were left unstimulated (control).
Phosphorylated Statl levels was assessed by flow cytometry as described above.
Results
Type 1 interferons (1FN-a/f3) and the more recently identified type Ill 1FNs
(1FN-X)
function as the first line of defense against virus infection, and regulate
the development
of both innate and adaptive immune responses. Type TIT IFNs were originally
identified
as a novel ligand-receptor system acting in parallel with type T TFNs, but
subsequent
studies have provided increasing evidence for distinct roles for each IFN
family.
The inventors aimed to evaluate the effects of type land type Ill interferons
on both innate
(antiviral) and adaptive immune response (CD4+ T cell proliferation).
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Antiviral activities of types I and III
The ability of IFN type I and III to induce the expression of interferon-
stimulated genes
(ISGs) was analyzed by qPCR.
Briefly, the antiviral activity of type I and III was tested in HepG2 cells
treated with
5 IFN-2a, IFN2d, IFN22. IFNA3 or IFN24 for 4 hours. Then the induction of the
well-known interferon-stimulated genes (ISGs) MX1, IFIT1 and OASL was
monitored
by qPCR.
As shown in Figure 1A, all five interferons clearly induced all three ISGs.
Since the investigated ISGs are functionally related to an antiviral defense,
the inventors
10 further evaluate the capacity of both IFN to protect HepG2 cells
from EMCV-induced
cytopathogcnic effect.
Briefly, cells were seeded in a 96-well microtiter plate and treated with the
indicated
amount of IFNs for 24 h and then challenged with EMCV for 20 h. Cell survival
was
measured by an MTT coloring assay.
15 As shown in Figure 1B, IFN type 111 and IFN- a2a have intrinsic
cellular antiviral activity
and are able to fully protect HepG2 cells challenged with EMCV.
Anti-proliferative activity of type I and type III interferons against CD4+ T
cells
proliferation
The effect of IFN-type I and IFN type III on CD4+ T cells proliferation in
response either
20 to polyclonal or to allogeneic stimulation was evaluated in a
mixed lymphocyte reaction
(MLR) assay.
Briefly, CFSE labelled CD4+ T cells were first stimulated with poly I:C
matured
allogeneic dendritic cells in presence of different dose of 1FNs. At 5 days
post activation,
the CFSE fluorescence dilution was analyzed.
25 As shown in Figure 2, IFN-a2a inhibits the proliferation of
stimulated CD4+ T cells,
while IFN type III exhibits no ability to suppress their proliferation. Of
note, when the
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MLR was performed in the presence of anti-interferon type I receptor antibody,
CD4+
T cells exhibit a greater proliferation. Thus, IFN-type I but not IFN type III
inhibit the
proliferation of allo-activated CD4+ T cells.
Moreover, the analysis of mRNA levels of the interferon-induced genes (ISG),
IFIT1,
MX1 and OASL in IFNs treated CD4+ T cells confirmed the lack or minimal
sensitivity
of CD4+ T cells to interferon type III.
Indeed, as shown in Figure 3, ISGs are induced only in CD4+ T cells stimulated
with
lFN-a2a. Thus, IFN-a2a but not IFN-type III induce the expression of ISGs in
CD4
T cells.
Because the Jak-STAT1/2 pathway being the major regulators of the
transcription of ISG,
the inventors have analyzed the phosphorylation levels of Statl proteins in
response to
lFN-type I, or interferon type III within CD4+T cells.
As shown in Figure 4, only IFN-a2a was able to stimulate the phosphorylation
of Stall
within CD4+ T cells. Therefore, IFN-a2a but not IFN-type TIT induces tyrosine
phosphorylation of STAT1 in CD4+ T cells.
Induction of chronic immune activation in presence of type land III
interferons.
Because chronic immune activation has been reasoned to be a significant
contributor to
disease progression in HIV-1-infected subjects, it is possible to monitor
disease
progression by measuring the expression of activation markers on CD4+ T cell
surface.
Thus, the inventors have evaluated, by flow cytometry, the capacity of both
1FNs to
increase the CD38 expression on stimulated CD4+ T cells.
As shown in Figure 5, only IFN-a2a was able to enhance the expression of CD38
on
stimulated CD4+ T cells.
Collectively, these ex vivo experiments show that while exhibiting anti-viral
activity, as
does IFN-a. interferon type III, by contrast to the immunosuppressive IFN-a
have no
effect on CD4+ T cell activation and proliferation. Indeed, interferon type
III do not inhibit
the initiation of the adaptative immune reaction as do TEN- a2a.
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In conclusion, while interferon type I and type III are induced by the same
viral
stimulating factors and exhibit similar signature profiles, their biological
activity appears
not redundant but rather complementary. Indeed, following viral infection,
during the
innate phase of the immune response, interferons type III exert their
antiviral effects in
mucosal sites whereas IFN-a act more systemically in the whole organism.
Furthermore,
the subsequent adaptive immune reaction is inhibited at its initiation level
by the
immunosuppressive effect of the IFN-a on activated CD4+ T cells.
Example 2: Critical pathogenic role of IFN-a and IFN-X in human HIV-1
infection
Material and Methods
Cryopreserved PBMCs were thawed in RPMI 1640 with 10% fetal bovine serum (FBS)
and washed in FACS buffer. Phenotypic staining was performed on 106 cells by
incubation with a viability marker (AmCyan live-dead kit from Invitrogen) and
with
antibodies conjugated to CD3, CD4, CD8, CD45RA, CCR7. Subsequently, cells were
washed, fixed with 4% parafortinaldehyde for 5 min, washed, and acquired with
an
AURORA cytometer (Cytek).
Peripheral Blood samples were obtained either from healthy donors through
Etablissement Francais du Sang (EFS, Paris, France) or from Elite controller
HIV-1
patients and chronically-HIV-infected patients pre and post combined ART
treatment.
Blood cells were collected using standard procedures. The study was performed
according to the Helsinki declaration, and the study protocol was reviewed and
approved
by the local Ethics Committee. All samples were de-identified prior to use in
this study.
Frozen serums were thawed at 4 C and centrifuged at 4000 G for 10 min at 4 C.
IFN-a
and IFN-X, (IL-28A) serum concentrations were measured using the high
sensitivity
Single-Molecule Array (Simoa0) technology (Digital ELISA technology)
(Quanterix)
according to the manufacturer's instructions.
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Results
Comparison of central memory (CM) CD8-F T cell distributions in HIV-I-infected
subjects
In study of chronically HIV-1-infected subjects, the following groups were
studied:
(i) elite controllers (EC) who naturally suppress HIV-1 in the absence of
combined
antiretroviral therapy treatment (c-ART)
(ii) non-controllers before (pre-cART) and after cART (post-cART)
treatment, and
(iii) a cohort of age-matched healthy donor (HD) subjects.
The relative frequencies of the CM populations within the CD8+ T cell
compartments
were evaluated in each of the subject groups.
The gating strategy to define this subset is the following. Briefly, singlet
cells were
defined, followed by gating on lymphocytes and live cells. Among the live
cells, CD3+
T lymphocytes were identified, followed by the definition of CD8+
subpopulations.
Subsequently, the expression of CD45RA and CCR7 was analyzed in the CD8+
T lymphocytes. Central memory T cells (TCM) are CD45RA- CCR7+.
Figure 6A shows that the level of CM CD8+ cells was significantly lower in
non-controllers before cART than in other groups. Moreover, combined
antiretroviral
therapy (cART) results in increase of CM CD8+ cells.
Comparison of serum IFN-a levels in HIV-I-infected subjects
Serum levels of 1FN-a was measured in the 4 groups. Figure 6B shows that the
non-controller patients have significantly increased serum IFN-a levels before
treatment
compared with after treatment.
IFN-a inversely correlates with the percentage of CM CD8 cells in HIV-infected
patients without treatment
In the population of HIV infected patients (EC pre-cART patients), the
inventors explored
the potential correlations between the level of CM CD8- cells and the scrum
1FN-a levels.
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In this study, there was a significant negative correlation between the
frequency of CM
CD8- cells and serum IFN-a levels (spearman correlation r = -0,667; p <
0.005).
This reflects the critical pathogenic effect of IFN-a on T cell proliferation
in secondary
organs ( see Figure 6C).
Comparison of serum IFN-2 levels in HIV-]-infected subjects
Serum from healthy control donors and HIV-1 infected patients, before and
after cART
treatment, originating from two distinct institutes were assayed by Simoa0 for
IFN-a and
IFN-X2 proteins.
Before cART treatment, serum levels of both IFN-a and IFN-X were higher in
patients
versus controls. After cART treatment, the levels of IFN-a had decreased but
the levels
of IFN-X remained unchanged (Figure 7A) or increased slightly (Figure 7B).
Conclusion
Latent HIV-1 reservoir represents the main obstacle in achieving sustained
virologic
remission in cART treated HIV-I infected patients following ART treatment
interruption.
This is due to two opposing biologic processes occurring in parallel in
patients under
cART:
1- On the one hand, cART promotes an inhibition of the viral replication
triggered by the HIV-1 proviral DNA present in activated peripheral and
mucosal reservoir cells. It results from this process a minimal expression of
viral particles in the body fluid (<40 copies/ml).
2- On the other hand, the second process is due to the production of type I
IFN-a and type III IFN-k locally, induced by viral replication occurring in
these still infected peripheral and mucosal cells, which contain latent
integrated HIV proviruses.
By their antiviral effects, these interferons locally reduce the ongoing viral
replication
occurring in these reservoir cells, which limits the local propagation of the
virus to nearby
cells but still maintains the reservoir of infected cells.
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The local production of IFN-a and IFN-k by peripheral and mucosal cells, which
are still
infected and replicating the virus, is confirmed by the presence in the serum
of post-cART
patients of substantial concentrations of these antiviral cytokines (Figure
7).
Following the blocking of the peripheral and mucosal antiviral interferons,
which hinder
5 expression of viral replication in reservoir cells, the HIV- 1 proviral
DNA present in these
reservoir cells may fully replicate viruses. In turn, cART treatment, which
controls viral
replication, may reduce and progressively eliminate these peripheral and
mucosal cellular
reservoirs.
Example 3: Clinical protocol for AIDS treatment in human patients
10 Study design I
Study eligibility criteria includes patients having ongoing ART (three drugs)
with
plasma H1V-1 RNA levels <40 copies/ml as well as a CD4+ T cell count > 500
cells/pi
(arid optionally < 500 cells/pi). Patients receive infusions of neutralizing
monoclonal
antibodies anti-IFN type I and type TIT receptors at 2-week intervals in
parallel with their
15 ongoing daily cART therapy (Figure 8). Plasma HIV-1 RNA levels, size of
the reservoir
and CD4+ T cell counts are monitored every 2 weeks, after collection of blood
samples,
1 day before each mAbs infusion.
Study design 2
Study eligibility criteria includes patients having ongoing ART (three drugs)
with
20 plasma HIV-1 RNA levels <40 copies/ml as well as a CD4+ T cell count >
500 cells/pi
(and optionally < 500 cells/pl). Patients receive infusions of anti-IFN-a,
anti-IFN-k
monoclonal antibodies and optionally anti-IFN-I3 monoclonal antibodies at 2-
week
intervals in parallel with their ongoing daily cART therapy (Figure 8). Plasma
HIV-1
RNA levels, size of the reservoir and CD4+ T cell counts are monitored every 2
weeks,
25 after collection of blood samples, 1 day before each mAbs infusion.
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Plasma HIV-] RNA Levels
HIV-1 RNA levels are determined using the Roche COBAS AmpliPrep/COBAS TaqMan
HIV-1 Assay (version 2.0) or the Roche cobas HIV-1 quantitative nucleic acid
test
(cobas 6800), which quantitate HIV-1 RNA over a range of 2x101 to lx107
copies/ml.
Quantitative Viral Outgrowth Assay
Size of the HIV reservoir is assessed with Quantitative Viral Outgrowth Assay
(QVOA)
as previously described (Huang et al., 2018, J Clin Invest 128:876-889).
Briefly, isolated
CD4+ T cells are plated out in serial dilutions (2, 1, 0.5, 0.2, and 0.1
million per well)
into 12 wells in a 24-well plate with phytohemagglutinin (PHA; 2 ittg/m1) and
irradiated
HIV-negative donor PBMC (2x106 cells/well) to reactivate the infected cells.
After 2 days
of culture, MOLT-4 CCR5 cells (2x106 cells/well) are added to amplify the HIV.
After 2 weeks of culture, p24 antigen is quantified in the culture
supernatant, using an
HIV p24 antigen enzyme-linked immunosorbent assay (ELIS A) kit (Perkin-Elmer,
Hopkinton, MA), and estimated frequencies of cells with replication-competent
HIV-1
are calculated using limiting dilution analysis.
CD4+ T-cell counts
CD4+ T-cell counts are determined by a clinical flow cytometry assay.
Treatment interruption
Monoclonal antibodies infusion is stopped when no cells with replication-
competent
HIV-1 are detected following 2-3 consecutive limiting dilution virus outgrowth
assays
(VOA). Then, ART treatment is stopped when no cells with replication-competent
HIV-1 are detected with 3-4 consecutive limiting dilution virus outgrowth
assays.
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Example 4: Humanized HIV-infected mice produce type I and type III
interferons.
Material and Methods
Animals
Animal experiments were carried out with female NOD/Shi-scid/1L-2Rynull (NOG)
immunodeficient mouse strain. Mice were humanized using hematopoietic stem
cells
(CD34+) isolated from human cord blood.
Humanized animal model
Humanized mice were intraperitoneally injected with HIV-NL4.3, and mock-
infected
mice were subsequently injected with an equal volume of the vehicle. Blood and
spleen
were removed from infected and non-infected humanized NCG mice at day 2 post-
infection, when the virus load has reached a plateau at day 10 and at day 30.
11-7\1-Al and IFN-A3 mRNA isolation and real-time PCI? analysis.
RNA was extracted from cells. Samples were DNase treated and then converted
into
cDNA (reverse transcribed) using the SuperScript III First-Strand Synthesis
system
(Invitrogen). The cDNA was used at 1:10, and expression of IFN-a, IFN-21, and
IFN-
X3were analyzed using the SYBR Green PCR-based protocol with the ABI Prism
7000
Sequence Detection System (Applied Biosystems). Gene-specific forward and
reverse
primers were designed using Primer Express software 1.5 (Applied Biosy stems).
The
housekeeping RPS14 gene was used to normalize cDNA across samples. The
conditions
for PCR were as follows: 10 min at 95 C, then 40 cycles of amplification at 95
C for 15
s and 60 s at 60 C, and finally 15 s at 95 C, 30 s at 60 C, and 15 s at 95 C.
Ct was
normalized to RPS14 (ACt = Ct sample ¨ Ct RPS14). Statistical analyses were
performed
by the 2¨AACT method.
Results
We studied the kinetics and localization of type I and type III IFN appearance
in HIV-
infected humanized mice. Mice were sacrificed on the day of infection and 10
and 35
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days after infection. PBMCs and splenocytes were recovered and the presence of
IFN-a,
lFN-X3, and IFN-k1 mRNA was assessed by RT-qPCR. Figure 9 shows that both
types
of IFN were produced after infection. IFN-a was most notably present in PBMCs,
whereas IFN-k3 and IFN-k1 were present in splenocytes.
Example 5: Activated CD4+ T cells express IFN-A, Receptor.
Material and Methods
CD4+ T cell purification and culture
CD4+ T cells were purified from peripheral blood mononuclear cells (PBMCs)
obtained
from anonymous healthy blood donors (EFS). Ficoll (Ficoll Hystopaque; Sigma)
density
centrifugation was performed as per the manufacturer's instructions, and CD4+
cells were
negatively selected using magnetic beads (CD4+ T-cell isolation kit I;
Miltenyi Biotec).
Purity was assessed following cell isolation by staining with anti-CD4-Alexa
Fluor 700
(RPA-T4) and all samples were >97% CD4+ by flow cytometry.
CD4+ T cells were cultured in RPMI 1640 supplemented with 10% FBS (Gibco), 100
IU
penicillin, 100 iug/mL streptomycin, 0.1 M Hepes, 2 mM L-glutamine, and/or
recombinant human IL-2. Cells were maintained at 37 C in a 5% CO2 humidified
incubator.
CD4+ T cells were co-stimulated through CD3 and CD28 using plate-bound
antibodies
for 3 days or no simulation. Anti-CD3 (OKT3) was used at 1 [tg/mL (BioLegend,
San
Diego, CA) and anti-CD28 (CD28.2) at 1 [tg/mL (BioLegend, San Diego, CA).
IL-28RA, IL-10RB, IFIH1 and IFI27 InRNA isolation and real-time PCR analysis,
RNA was extracted from cells. Samples were DNase treated and then converted
into
cDNA (reverse transcribed) using the SuperScript III First-Strand Synthesis
system
(Invitrogen). The cDNA was used at 1:10, and expression of IL-28RA, IL- 'ORB,
IFIH1
and IFI27 were analyzed using the SYBR Green PCR-based protocol with the ABI
Prism
7000 Sequence Detection System (Applied Bio systems). Gene-specific forward
and
reverse primers were designed using Primer Express software 1.5 (Applied
Biosystems).
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The housekeeping RPS14 gene was used to normalize cDNA across samples. The
conditions for PCR were as follows: 10 min at 95 C, then 40 cycles of
amplification at
95 C for 15 s and 60 s at 60 C, and finally 15 s at 95 C, 30 s at 60 C, and 15
s at 95 C.
Ct was normalized to RPS14 (ACt = Ct sample ¨ Ct RPS14). Statistical analyses
were
performed by the 2¨AACT method.
Gene expression analysis of interferon stimulating genes IFIH1 and IFI27
Cells were treated with 100 IU/ml of type I IFN (IFN¨a) or 10 ng/ml of type
III IFN
(IFN-23). Total RNA was isolated at 16 hours post-treatment.
Flow Cytometry Analysis.
For IFN-X3 binding assay to quantify the presence of IL-28RA on CD4+ T cells,
cells
were treated with or without His-tagged IFN-X3 (R&D Systems) diluted in PBS
containing 1 % BSA on ice for 60 min at indicated doses. Cells were then
washed and
stained with anti-His PE (Miltenyi Biotec) for 40 min in the dark and a
viability dye. The
MFIs of IFN-X3 binding on CD4+ T cells were determined by flow cytometry in
living
cells as follows: MFI (His-tagged IFN-23 + anti-His PE) ¨ MFI (anti-His PE).
Results
While IFN type I receptors (IFNAR1/2) are known to be ubiquitous and expressed
on
almost all cell types, IFNLR1 is described to be more restricted and to be
highly expressed
on epithelial cells, neutrophils and dendritic cells. Some teams reported that
CD4+ T cells
were sensitive to type III IFN, others indicate that they were not. These
discordant results
are due to the different biological contexts when the analysis of the
expression of the
receptor was performed.
We assessed the ability of CD4+ T cells to express IFNLR according to their
level of
activation. We purified primary CD4+ T cells and stimulated them without or
with anti-
CD3 and anti-CD28 antibodies in the presence of IL-2. After three days of
culture, we
studied the presence of IL28RA and IL-10RB transcripts (these two molecules
constituting IFNLR), the ability of the cells to bind IFN-23 and to produce
the IFIH1 and
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IF127 mRNA interferon stimulated genes (ISO) after their culture in the
presence of IFN-
X3. Figure 10 shows that while purified CD4+ T cells cultured in the presence
of IL-2
alone expressed basal levels of IL-28RA mRNA and IFNLR, the 3-day-CD3/CD28/IL-
2-stimulated cells displayed high level of IFNLR, at mRNA and protein levels.
The IFIH1
5 and IF127 mRNA detection in 1FN-X3-treated stimulated CD4 T cells confirmed
the
presence of a functional IFNLR at their surface.
Example 6: IFN-13 inhibits HIV-1 infection of activated 034+ T cells.
Material and Methods
CD4 T cell purification and culture
10 CD4+ T cells were purified and cultured as described in Example
5.
Reagents
IFN-X3 and anti-IFNAR2 neutralizing antibody were purchased from Bio-Techne
and anti-IL28RA (IFNLR1) neutralizing antibody from PBL assays science.
Production of Viral Stocks
15 HIV NL4.3 was obtained from the AIDS Research and Reference
Reagent Program. Viral
stocks were generated by transfection of HEK 293T with polyethylenimine
(Polysciences). Two days after transfection, culture supernatants were
collected, clarified
at 441 x g for 5 min, and filtered (0.45 km).
HIV Infection Experiments
20 CD4+ T cells were co-stimulated through CD3 and CD28 using plate-
bound antibodies
for 3 days or no simulation. Anti-CD3 (OKT3) was used at 1 p..g/mL (BioLegend,
San
Diego, CA) and anti-CD28 (CD28.2) at 1 kg/mL (BioLegend, San Diego, CA).
Stimulated CD4+ T cell HIV-1 infections occurred after 3 days of CD3/CD28
stimulation.
Infections with HIV-NL4.3 were performed overnight in the presence of
polybrene
25 (2 kg/mL), and fresh media were replaced. The frequency of
infected cells was
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determined by intracellular HIV p24 by FACS. When indicated, IFN-c, IFN-23,
anti-
IFNAR2 and anti-IL-28RA neutralizing antibodies were added 24 hours before
infection
and for an additional 5 days in culture after infection.
Results
We then investigated the ability of type III IFNs to prevent HIV-1 infection
of activated
CD4+ T cells, using as control type I IFN known to strongly inhibits HIV-1
infection.
Figure 11 shows that both types of IFN inhibited infection of activated CD4 T
cells,
with type I IFN inhibition being slightly more important. Interestingly,
stimulation of
CD4+ T cells by both IFNs simultaneously increased the blockage of infection.
Example 7: Sequential treatment with two latency-reversing agents, DNA
methylation inhibitor 5-AzadC and HDACI MS-275, induce IFN-). Receptor
expression on CD4+ T cells.
Material and Methods
CD4+ T cell purification and culture
CD4+ T cells were purified, cultured and co-stimulated as described in Example
5.
Jurkat cell culture
The human cell line Jurkat (J-Lat) clone 10.6, was obtained from AIDS Reagent
Program,
National Institutes of Health (Germantown, MD). This cell line was grown in
complete
culture medium, as the primary cells, and maintained at 37 C in a 5% CO2
incubator. J-
Lat clone 10.6 harbors a HIV provirus containing the GFP open reading frame
(ORF)
instead of nef and a frameshift mutation in env.
Reagents
5-AzadC was purchased from Sigma Aldrich, MS-275 from Enzo Life Sciences and
FN-
X3 from Bio-Techne.
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LRA treatment of stimulated CD4+ T cells
Stimulated CD4+ T cells were submitted to a 48-h 5-AzadC pretreatment followed
by a
24-h MS-275 induction, corresponding to a total 72-h 5-AzadC treatment.
HIV reactivation in vitro in latently infected J-Lat clone 10.6.
HIV reactivation was performed by a 48-h 5-AzadC pretreatment followed by a 24-
h MS-
275 induction, corresponding to a total 72-h 5-AzadC treatment. Reactivation
of HIV was
monitored as the percentage of living GFP-positive cells, according to forward
and side
laser light scatter flow cytometry analysis in a FACS ARIA3 flow cytometer (BD
Biosciences). The data were analyzed using FlowIo software.
IL-28RA, IFIHI and IFI27 mRNA isolation and real-time PCR analysis.
RNA was extracted from cells. Samples were DNase treated and then converted
into
cDNA (reverse transcribed) using the SuperScript 111 First-Strand Synthesis
system
(Invitrogen). The cDNA was used at 1:10, and expression of IL-28RA, IFIH1 and
IFI27
were analyzed using the SYBR Green PCR-based protocol with the ABI Prism 7000
Sequence Detection System (Applied Biosystems). Gene-specific forward and
reverse
primers were designed using Primer Express software 1.5 (Applied Biosy stems).
The
housekeeping RPS14 gene was used to normalize cDNA across samples. The
conditions
for PCR were as follows: 10 min at 95 C, then 40 cycles of amplification at 95
C for 15
s and 60 s at 60 C, and finally 15 s at 95 C, 30 s at 60 C, and 15 s at 95 C.
Ct was
normalized to RPS14 (ACt = Ct sample ¨ Ct RPS14). Statistical analyses were
performed
by the 2¨AACT method.
Flow Cytometry Analysis.
For IFN-X3 binding assay to quantify the presence of IL-28RA on CD4+ T cells,
cells
were treated with or without His-tagged IFN-X3 (R&D Systems) diluted in PBS
containing 1% BSA on ice for 60 min at indicated doses. Cells were then washed
and
stained with anti-His PE (Miltenyi Biotec) for 40 min in the dark and a
viability dye. The
MFIs of IFN-X3 binding on CD4+ T cells were determined by flow cytometry in
living
cells as follows: MET (His-tagged 1FN-X.3 + anti-His PE) ¨ MFI (anti-His PE).
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Results
Reactivation of HIV gene expression in latently infected cells together with
an efficient
cART has been proposed as an adjuvant therapy aimed at eliminating/decreasing
the
reservoir size. So far, none of the LRAs that have been tested have been
successful in
eradicating HIV-1 latent reservoir.
We investigated whether these molecules could influence the IFNLR expression
in CD4
T cells. Briefly, stimulated CD4 T cells and latently infected J-Lat 10.6
cells (cell lines
that harbor an HIV provirus containing the Green Fluorescent Protein (GFP) ORF
instead
of nef and a frameshift mutation in env) were treated sequentially with two
latency-
reversing agents, DNA methylation inhibitor 5-AzadC and HDACI MS-275, a
combination of molecule known to reactivate HIV. After a 72-h treatment with 5-
AzadC
and MS-275 (MS-275 having been added during the last 24 hours), we studied the
presence of IL28RA (IFNLR1) transcript in both cells, their ability to bind
IFN-X3 and
to produce the IFIH1 and IFI27 mRNA interferon stimulated genes (ISG) after
their
culture in the presence of IFN-X3. Figure 12 shows that the sequential
association of
these two molecules increased IFNLR mRNA and protein levels in both T cells.
In
addition, their stimulation with IFN-2L3 led to the induction of the IFIH1 and
1F127 ISG.
Collectively these data indicate that this LRA combination treatment could
make the cells
sensitive to type III interferon.
Example 8: The NtRTI TDF enhances the production of IFN-X3 by HT-29 cells.
Material and Methods
1-IT-29 cell culture
Human colon adenocarcinoma HT-29 cells (ATCC, HTB-38) were cultured in 25 cm2
culture flasks in McCoy's 5A medium supplemented with 10% heat-inactivated FCS
(Gibco), penicillin (100 U/mL) and streptomycin (100 pg/mL).
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TDF treatment of HT-29 cells
HT-29 cells were incubated with dimethyl sulfoxide (DMSO) or tenofovir
disoproxil
fumarate (TDF, 25 laM) for 48 hours.
IFN-113 analysis of supernatants
The levels of IFN-2,3 were assayed using an ELISA kit according to the
manufacturer's
instructions.
Results
Since patients undergoing antiretroviral treatment have a persistent type
1/type Ill
interferon signature, we looked at the effects of certain antiviral molecules
on type III
1FN production. Since HIV infection occurs at the gastrointestinal level and
antiviral
treatments are taken orally, we looked at the effect of Tenofovir disoproxil
fumarate
(TDF), a nucleotide reverse transcriptase inhibitor (NRT1) of HIV commonly
used, on
intestinal epithelial HT-29 cells. Figure 13 shows that lEN-X3 was detected in
the culture
supernatants of HT-29 cells treated with TDF.
Example 9: IFN-13 secreted by TDF-treated HT-29 cells impairs CD4+ T cells
infection.
Material and Methods
CD4+ T cell purification and culture
CD4+ T cells were purified and cultured as described in Example 5.
HT-29 cell culture
Human colon adenocarcinoma HT-29 cells (ATCC, HTB-38) were cultured as
described
in Example 8.
Reagents
Anti-IL28RA neutralizing antibody was purchased from PBL assays science.
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Production of Viral Stocks
HIV NL4.3 was obtained from the AIDS Research and Reference Reagent Program.
Viral
stocks were generated by transfection of HEK 293T with polyethylenimine
(Polysciences). Two days after transfection, culture supernatants were
collected, clarified
5 at 441 x g for 5 min, and filtered (0.45 um).
Transwell co-culture model for assessing inhibition of HIV infection in
stimulated CD4+
T cells by IFN-23 released by TDF-treated HT-29 cells.
Experiments were performed using 12-transwell chambers with a polycarbonate
filter
(0.2 pm pore size). HT-29 were grown until subconfluence in the lower chamber.
Then
10 HT-29 cells were treated with tenofovir disoproxil fumarate (TDF). After
24h of TDF
treatment, 3-day-CD3/CD28/IL2- stimulated CD4+ T cells were seeded in presence
of IL-
2 in the upper chamber, which is then placed in the culture well so as to be
immersed in
the culture medium of the HT-29 cells. When indicated, the CD4+ T cells were
pre-treated
with an IFNLR1 (IL28RA) neutralizing antibody. After 24h of co-culture, CD4+ T
cells
15 were isolated, infected with HIV NL4.3 and then placed back into the co-
culture for an
additional 5 days of culture. The frequency of infected cells was determined
by
intracellular HIV p24 by FACS.
Flow Cytornetry Analysis.
For the HIV p24 intracellular staining, cells were fixed and permeabilized
with the BD
20 fix and perm kit and stained with Kc-57-RD1 (1:100; Beckam-Coulter).
Results
In order to get closer to an in vivo context, we developed a Transwell culture
assay to
evaluate the IFN-X3 antiviral activity. The test was performed in 12-well
Transwell plates
with stimulated CD4+ T cells at sufficient concentrations (Figure 14).
Briefly, HT-29
25 cells were cultured in the bottom chamber of Transwell plates. When the
culture was
subconfluent, Tenofovir disoproxil fumarate (TDF) was added. After 24 hours of
stimulation, activated CD4 T cells were deposited in the insert which was then
placed in
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the culture well. When indicated, the CD4+ T cells were pre-treated with an
IFNLR1
(IL28RA) neutralizing antibody. After 24 hours of co-culture, the cells in the
insert were
harvested, infected and then placed back into the co-culture for an additional
5 days of
culture. The frequency of infected cells was determined by intracellular HIV
p24 by
FACS. In this context, Figure 15 shows that the IFN-X3 released by TDF-treated
HT-29
cells was able to impair HIV-1 CD4+ infection.
Example 10: IFN-13 released by TDF-treated HT-29 cells inhibit in vitro HIV
reactivation in latently infected J-Lat clone 10.6.
Material and Methods
Reagents
5-AzadC was purchased from Sigma Aldrich; MS-275 from Enzo Life Sciences; and
IFN-
X3, from Bio-Techne. Anti-IL28RA neutralizing antibody was purchased from PBL
assays science.
HT-29 cell culture
Human colon adenocarcinoma HT-29 cells (ATCC, HTB-38) were cultured as
described
in Example 8.
Jurkat cell culture
The human cell line Jurkat (J-Lat) clone 10.6 was cultured as described in
Example 8.
Transwell co-culture model for assessing the inhibition of HIV latency
reversal in latently
infected J-Lat cells by IFN-L3 released by TDF-treated HT-29 cells.
Transwell cultures (12 wells) with confluent HT-29 monolayers were used for co-
culture
with J-LAT 10.6 cells. HT-29 were grown until subconfluence on Transwell
bottom.
Then HT-29 cells were treated with Tenofovir disoproxil fumarate (TDF). After
24h of
TDF treatment. J-LAT 10.6 cells pretreated with 5-AzadC and MS-275 for 72h
were
seeded on Transwell filters. When indicated, anti-IFNLR1 (IL28RA) neutralizing
Abs
were added to the culture 2 hours before the J-Lat 10.6 cells seeding. After
48 h of co-
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culture, HIV reactivation was monitored as the percentage of living GFP-
positive cells,
according to forward and side laser light scatter flow cytometry analysis in a
FACS
ARIA3 flow cytometer (BD Biosciences). The data were analyzed using FlowJo
software.
Results
We finally determined whether type III interferon could impair HIV-1 latency
reversal.
We used the Transwell culture assay we have set up to evaluate the IFN-X3
antiviral
activity. We placed the HT-29 cells in the bottom chamber of Transwell plates
and the J-
Lat cells pretreated with 5-AzadC and MS-275 in the insert (Figure 16).
Figure 17 shows that IFN-X3 released by TDF-treated HT-29 cells inhibited HIV
latency
reversal.
Example H: Pre-Clinical protocol for HIV-1 reservoirs elimination in HIV-1
infected humanized mice.
Study design
Two humanized mouse models are used, BLT mice and NCG mice. Humanized BLT
mice with > 50% of circulating human CD45+ cells at 24 weeks post-
transplantation and
humanized NCG mice with 20% of circulating human CD45+ cells at 24 weeks post-
transplantation are infected with HIV-1 through retro-orbital (BLT mice) or
intraperitoneal injection (NCG mice). Hu-mice infected with HIV-1 for 7-9
weeks are
treated with cART. The study is structured into two arms (Figure 18). The mice
are
injected with a-IFNAR1 mAb and a-IFNLR1 mAb (experimental group) or with
corresponding isotype mAbs (control group) twice a week starting from week 4
after
cART. cART is maintained for an additional 2.5 weeks after the last antibody
treatment.
At the end of the cART treatment, animals are sacrificed. Some animals are
kept after
cART has been discontinued to measure time to viral detectability after
treatment
interruption. These groups of "viral rebound" are monitored for another three
to six weeks
or longer if they remain undetectable in plasma viral loads. Monitoring of
viral load upon
HIV-1 infection and under cART treatment is performed every two weeks.
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Plasma HIV-] RNA Levels
HIV-1 RNA levels are determined using the Roche COBAS AmpliPrep/COBAS TaqMan
HIV-1 Assay (version 2.0) or the Roche cobas HIV-1 quantitative nucleic acid
test
(cobas 6800), which quantitate HIV-1 RNA over a range of 2x101 to lx 107
copies/ml.
Cell-associated HIV-1 DNA detection.
To measure total cell-associated HIV-1 DNA, nucleic acid is extracted from
spleen and
bone marrow cells using the DNeasy mini kit (Qiagen). HIV-1 DNA is quantified
by real-
time PCR. DNA from serial dilutions of ACH2 cells, which contain 1 copy of HIV
genome in each cell, is used to generate a standard curve.
Cell-associated HIV-] RNA detection.
To measure total cell-associated HIV-1 RNA, nucleic acid is extracted from
spleen or
bone marrow cells using the RNeasy plus mini kit (Qiagen). HIV-1 RNA is
detected as
described above. The HIV-1 RNA expression levels are normalized to human CD4
mRNA controls, and the result is calculated as fold change in gene expression.
Quantitative Viral Outgrowth Assay
Size of the HIV reservoir is assessed with Quantitative Viral Outgrowth Assay
(QVOA)
as previously described (Huang et al., 2018, J Clin Invest 128:876-889).
Briefly, isolated
CD4+ T cells are plated out in serial dilutions (2, 1, 0.5, 0.2, and 0.1
million per well)
into 12 wells in a 24-well plate with phytohemagglutinin (PHA; 2 lig/m1) and
irradiated
HIV-negative donor PBMC (2x106 cells/well) to reactivate the infected cells.
After 2 days
of culture, MOLT-4 CCR5 cells (2x106 cells/well) are added to amplify the HIV.
After 2 weeks of culture, p24 antigen is quantified in the culture
supernatant, using an
HIV p24 antigen enzyme-linked inamunosorbent assay (ELISA) kit (Perkin-Elmer,
Hopkinton, MA), and estimated frequencies of cells with replication-competent
HIV-1
are calculated using limiting dilution analysis.
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Example 12: Discussion
Lymphoid reservoir formations present before cART in infected patients
HIV infection in patients is maintained by some lymphoid formations called
reservoirs.
These formations are found anatomically on the intestinal or vaginal mucous
membranes
initially (during the primary infection), and then within the peripheral lymph
nodes or
spleens. These lymphoid formations contain virions in their circulating lymph
and host
three categories of CD4+ T cells:
1) infected CD4+ T cells that are immune-activated by specific antigenic
stimulation,
2) infected CD4+ T cells that are immune-resting. These cells are resting
although
they carry the proviral DNA integrated into their genome. These cells do not
synthesize
viral proteins until they are immune-stimulated.
3) healthy CD4+ T cells that are not carrying the proviral DNA and are resting
while awaiting their specific antigenic stimulation.
These formations actually maintain virus synthesis thanks to infected CD4+ T
cells
undergoing specific immune activation induced by their antigen. These cells
produce
stromal lymph virions and circulating blood virions and may spread the viral
infection to
other lymphoid formations. These latter formations in turn become reservoir
formations.
What happens under cART within these reservoir formations which contain the
three
categories of CD4+ T cells?
The behavior of these three categories of CD4 T cells is different during cART
treatment.
Their behavior is determined by the combined, and sometimes antagonistic,
biological
effect of three factors:
1) the presence of anti-viral messengers, namely mucosal type III interferon
and
systemic type I interferon induced by viral particles.
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2) pharmacological treatments (cART) which block any synthesis of viral
proteins
and virion production.
3) interferon-stimulated genes (ISGs) maintaining viral latency: these are a
set of
molecules that inhibit the transcription of HIV genes when they are integrated
into their
5 DNA.
Mucosal type III and systemic type I interferons (IFNs) represent the first
natural anti-
viral defense blocking the integration of proviral DNA in target CD4+ T cells
that carry
HIV receptors. These IFN messengers, present in the stromal lymph of the
lymphoid
formations, act in a paracrine and dose-dependent manner in neighboring target
CD4+ T
10 cells that carry their specific type I and type III receptors.
Their action induces the
expression of ISGs which inhibit the synthesis of viral proteins, such as
ISG15.
It should be noted that CD4+ T cells constitutively express type I IFN
receptors and
inducibly express type III IFN receptors. Activation of these receptors by
IFNs prevents
the expression of virions within CD4+ T cells by blocking viral protein
synthesis and
15 virion production. Thus, the infected resting cells in the
lymphoid reservoir formations,
that have the proviral DNA integrated into their genome, as well as the
healthy uninfected
resting cells are protected, do not synthesize viral proteins and do not
produce viruses.
On the other hand, infected cells of these lymphoid formations, which are
immune-
activated by their antigenic stimulation and which already produce the
virions, are not
20 sensitive to the IFNs action, but they produce the infectious
virions.
How does each of these three categories of CD4+ T cells evolve within the
mucosal and
peripheral lymphoid reservoir formations under cART?
1) HIV-infected and immune-activated CD4+ T cells (category 1) maintain their
viral production and die by apoptosis. However, before disappearing, the
virions they
25 produce induce the production of mucosa' type III antiviral IFNs in the
mucous
membranes and of systemic type I IFNs which protect the infected or non-
infected resting
CD4+ T cells (category 2 and 3) in a paracrine and dose-dependent manner.
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2) Infected CD4+ T cells which carry the integrated proviral DNA, but are not
immune-activated by their specific stimulation within the lymphoid foci, are
protected
from viral production by the production of ISGs induced by type III and type I
IFNs.
Indeed, the activation of type I and III IFN receptors that they express
activate the
ISGMLs responsible for the latency induced by the IFNs. IFNs are mainly
produced by
APCs (pDC, mDC and macrophages).
3) Uninfected cells not carrying proviral DNA (category 3) remain resting.
These
are healthy cells that are spared thanks to the paracrine action of IFNs
during their specific
antigenic stimulation, that behave like healthy and immune-activated CD4+ T
cells.
cART interruption
Stopping cART affects all three categories of CD4+ T cells of the reservoir
lymphoid
formations. It follows that:
1) The infected cells which were immune-activated during cART died after
producing the
virions which induce the secretion by APCs of type TTT and type I antiviral
IFNs in a
paracrine and dose-dependent manner.
2) The infected and non-immune-stimulated cells wait for their antigenic
stimulation to
be activated and release the ISGMLs of their latency. These cells will induce
the synthesis
of viral proteins and the production of virions circulating in the stromal
lymph of the
lymphoid foimations and then in the peripheral blood and will disseminate the
infection
to other so far spared lymphoid formations.
3) The uninfected and not yet stimulated cells will no longer be protected by
the IFNs and
during their specific stimulation will also produce viruses circulating in the
strornal
lymph. After production of virions, these cells will disappear by apoptosis,
which reduces
the supply of CD4+ T cells in the body.
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How to clear out lymphoid reservoir formations in patients under cART
optionally in
combination with antibodies neutralizing IFNalpha?
The invention proposes to combine these two treatments with agents blocking
mucosal
type III IFNs produced on the mucous membranes, such as neutralizing
antibodies. This
combination appears necessary to clear out the lymphoid reservoir formations
of their
vim s-producing cells that maintain the infection.
Indeed, our analysis of sera from patients under cART (still expressing a
certain virion
concentration) shows the presence of mucosal IFN-lambda acting on the mucous
membranes, which is the initial site of infection.
The gradual disappearance of infected CD4+ T cells by apoptosis, whether they
are
activated (category 1) or resting (categories 2 and 3), will allow eradication
of the HIV
infection. This elimination can be controlled by mucosal lambda IFNs in the
mucous
membranes and by systemic type I IFNs, which will prevent the decrease in CD4+
T cells
in the body. This decrease in CD4+ T cells actually decreases adaptive immune
capacity
and facilitates progression to disease. The viral load (number of virions/ml)
at the time of
the set point after the primary infection will be a major indicator of the
time required to
clear out the lymphoid reservoir formations to eradicate the infection.
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