7 HIV Persistence Workshop: fourth day PDF Print E-mail
Written by Alain Lafeuillade   
Sunday, 13 December 2015 22:07

7 HIV Persistence Workshop: fourth day

1-Monoclonal_antobodyThe last day of the workshop focused on new therapeutic approaches, in particular those using monoclonal antibodies.

It was also followed by a summary (Take Home Message) given by David Margolis

Finally it ended with the announcement of the 8th edition of the workshop in 2 years.

 

SESSION 9: New therapeutic approaches Part 1

 

Dr Mascola (Bethesda) talked about monoclonal antibodies showing that the new generation is 1,000 fold more potent than the first one.

However, no single monoclonal antibody (mAb) covers all viral isolates and due to Env variation, resistance to a single mAb will occur.

 

These new mAb have a lon half-life and can act for weeks or months after a single infusion.

He then concentrated his talk on 2 mAb, 3BNC117 and VRC01 that can induce a decrease of 1 - 2 log after infusion.

Other mAb are going into clinical trials.

Future improvements are:

-bispecific, bifunctional mAB

-increased in potency/breath


Bispecific antibodies target 2 different sites.

 

Bifunctional antibodies are DARTs and BITEs and target CD3 and HIV-1

 

 

These new mAb have a lon half-life and can act for weeks or months after a single infusion.

He then concentrated his talk on 2 mAb, 3BNC117 and VRC01 that can induce a decrease of 1 - 2 log after infusion.

Other mAb are going into clinical trials.

 

Future improvements are:

-bispecific, bifunctional mAB

-increased in potency/breath

 

Bispecific antibodies target 2 different sites.

Bifunctional antibodies are DARTs and BITEs and target CD3 and HIV-1

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Dr Nordstrom (Rockville) from Macrogenics talked further on DARTs (Dual Affinity Re Targetting) platform. They succeeded in producing more than 100 DARTs mire more than 40 specificities, to use in different illnesses.

 

Regarding HIV, they used anti-Env either from broadly neutralizing antibodies or non neutralizing Ab.

The A32 and 7B2 DART molecules were able to mediate potent CTL activity against HIV-1 infected resting CD4 T cells.

 

DART molecules reduced resting HIV reservoir Ex vivo

 

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There was no evidence of enhancement of virus spread with these antibodies in the system used.

 

In conclusion:

•Specifically redirect T cells to kill HIV-infected cells
–Dependent on target cell expression of env (low levels appear sufficient)
–Dependent on T cells as effectors (polyclonal, not MHC-restricted)
–EC50 in picomolar range
•Combinations with different Env specificities can be used to maximize activity against cells infected with diverse HIV isolates
•Capable of mediating reductions in resting and LRA-inducible HIV reservoirs from donors ex vivo
•Unlikely to pose a risk for enhancing the spreading of HIV infection
•Molecules in Fc-bearing format are active and have PK properties comparable to IgGs
•HIV x CD3 DART molecules are a promising therapeutic strategy for targeting HIV reservoirs – in vivo studies imminent

Dr Macedo (Salt Lake City) presented a TLR 2/7 agonist as a Latency Reversing Agent (LRA).

TLRs ligands leads to activation of the transcriptional factor NF-KB, required for HIV reactivation.

Recently they described that Pam3 is able to reactivate latent HIV in central memory CD4 T cells, without leading to T-cell activation

so, we would like to ask the question: is there another TLR2 ligand more potent than Pam3?

 

They decided to study CL572, which is a multi-PRR agonis.

CL572:

-induced higher reactivation than Pam3 CSK4 in a primary model of latency

-did not induce apoptosis and activation on resting CD4+ T cells from aviremic patients

 

CL572 exhibited synergy with Panobinostat, Romidepsin or JQ1 (BET inhibitor)

 

Dr Moron Lopez (Badalona) presented a project combining an immune stimulation with the early cART initiation.

Specifically, the immune-stimulation consisted in a prime with nonreplicating chimpanzee adenovirus type 63 and a boost with nonreplicating modified vaccinia virus Ankara.

The rationale for use ChAdV63 is to avoid humans responses against this viral vector, as it happened with the vaccines using AdV5 as viral vector.

The immunogen used was the HIVconsv, developed in Oxford by Thomas Hanke group, which encodes the 14 most conserved regions of the consensus HIV-1 Gag, Pol, Vif, and Env proteins from different clades of HIV-1.


PREVIOUS STUDIES in HIV negative individuals demonstrated:

1) Safety in prime/boost regimens using ChAdV63 and MVA viral vectors, and that this vaccine regimen
2) Elicited high frequency of both CD4 and CD8 T-cell responses (by IFNgamma and cytometry-IFNgamma, TNFalpha, IL2, CD107…), and
3) Induced T cells able to recognize and inhibit HIV-1 replication (by ELISA, viral inhibition)

 

The authors wanted to evaluate:

1) The safety and immunogenicity of ChAdV63.HIVconsv and MVA.HIVconsv vaccines, and

2) the effect of this prime/boost regimen on HIV reservoir and viral reactivation

in a cohort of early-treated HIV-1-infected subjects

Rationale for performing the trial in early-treated individualsà less immune exhaustion, better responsiveness to vaccination, limitation of the viral reservoir, best platform for future kick-kill strategies.

 

The BCN01 trial was a Phase I, non-randomized, multicentre study that included 48 individuals with recent infection (confirmed <6 months from infection) who initiated Tenofovir/Emtricitabine/Raltegravir within 1 week after diagnosis:

•24 were vaccinated:
• 12 boosted after 8 weeks: ‘short’ ARM, and
• 12 after 24 weeks: ‘long’ ARM, and
•24 were unvaccinated controls
They wanted to evaluate the induction of immune responses and the shift of CTL immuno-dominance pattern, by IFNgamma ELISPOT specific against HIVconsv and the other regions of HIV-1 clade B. AND in both arms, we observed an increase in the immune-responses in all the patients with the peak of immune-response between week 1 and 4 after MVA boosting, and also they observed that this induced responses were specific for HIVconsv because there was an increase in the focus.

The focus was a ratio between the specific immune-responses against HIVconsv and the HIV-specific responses against the other regions of HIV-1 clade B consensus.

THEREFORE, this prime/boost regimen shift the CTL immunodominance pattern from inespecific regions of HIV-1 to the specific conserved regions of HIVconsv.

They observed a significant decrease in all groups between the prime-baseline (wk24) and the weeks 58, 62 or 60 depending on the group.

This is what we can expected because all the groups were in the first year of antiretroviral treatment.

This decrease was not significantly higher in the vaccine groups compared to the control group, either in absolute quantification after a year of antiretroviral therapy, or in the decay, measured as the difference between week 24 and week 60 normalized to the reservoir at week 24.

 

The next step will be to combine this immune-stimulation and early cART initiation with a latency reversal agent, Romidepsin, to achieve a kick and kill approach.

 

Dr Giavedoni (San Antonio) presented data on the construction of nano-particles targeting CD4+ T cells for the delivery of SIV-specific RNA-guided Cas9 nucleases.


•Preliminary results show that several of their RNA-guided Cas9 nucleases can totally inhibit viral replication, as detected by SIV Gag p27 levels.
 
•The nature of such inhibition is being currently investigated.
 
•Protocell nanoparticles conjugated with α-CD4 IgG4 show great promise for facilitating delivery of therapeutic materials into CD4+ cells, based on our data showing exceptional preferential internalization.
 
•Currently they are loading vector encoded Cas9 into protocells to test whether these nanoparticles can effectively deliver biomaterials to infected CD4+ cells and reduce viral replication in vitro.
 
•Off-target effects need to be determined before moving into NHP.

Dr Felber (Frederick) focused on the "kill" aspect. She improved DNA vaccination in the SIV model by identifying 7 conserved regions of gag.
DNA vaccination + heterodimeric (het IL-15) IL15/IL 15 receptor alpha increased the number of effector CD8 cells in lymph nodes germinal centers.

Dr Luo (Winnipeg) showed that sequences surrounding the 12 protease cleavage sites can be targets for an HIV vaccine in a SIVmac251 model.
Dr Khalili (Philadelphia) studied the elimination of HIV-1 Genomes From Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing.
The aim is to eliminate proviral HIV DNA from cell genes but there are areas of concern:
-Off-target effects
-Continuous expression of Cas9…Immunogenicity
-Variability of HIV-1 DNA sequences in patients and quasispecies
-Delivery to viral reservoirs

 

SESSION 10: New therapeutic approaches Part 2


Dr Sekaly (Cleveland) talk was intitled "Anti-inflammatory cells and cytokines drive Homeostatic replacement and HIV reservoir".
•Several ICBs are associated with T cell dysfunction in chronic HIV infection (PD-1, CTLA-4, TIM-3, CD160) (Trautmann et al, NatMed 2006; Kaufmann et al, NatImm2007; Jones et al, JEM 2008, Peretzel al, PLoSPath 2012)
•Several have also been associated to the size of the HIV reservoir ( Chomont et al , Nat Med)
•In vivo , these molecules trigger different pathways
•Cells expressing these molecules shown show different functions
By inhibiting T cell activation negative regulators of of immune check points may actively maintain viral latency and identify reservoir cells during ART.

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PD1 maintains the reservoir by promoting homeostatic T cell proliferation with without replacement of T cells.
PD1+ LAG3- cells are mostly differentiated memory cells. They produce TGF-beta and inhibit homeostatic replacement of T cells.
PD-1 + cells include mostly highly differentiated mature T cells with high levels of inducible mRNA and which are engaged in a homeostatic proliferation.
 
LAG3 maintains the HIV reservoir by negative regulation of T-cell homeostasis:  LAG-3+ T cells contribute to the increase in reservoir by inhibiting the replacement of infected cells by newly produced T cells
LAG3+PD1- cells are mostly central memory cells.
LAG-3+ cells include mostly quiescent less differentiated mature T cells with low levels of inducible mRNA.

Diverse Tregs subsets have been characterized on the basis of their phenotype and capacity to produce specific cytokines:

Natural and inducible Treg subsets:

-Natural: CD25+FoxP3+

-Inducible: Tr1: IL-10-secreting, Lag3+ CD49b+, iTregs (Th3): TGF-β-secreting, CTLA-4+, nTregs : CD45RA+ Tregs

 

 Tregs and Tr1 cells play a critical role in establishment of HIV latency and CD4 senescence.

 

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IL-10 and TGF-B blockade have the potential to limit the establishment and maintenance of HIV reservoir

Genetically modified T cells (uninfectable) can contribute to replacement of bad cells with good cells

Dr Jerome (Seattle) spoke about ZFNs (Zinc Finger Nucleases) cleavage of different targets of HIV, for exemple targeting pol inhibits HIV replication.

Rare variants may retain replicative capacity after targeted mutagenesis; however, these remain susceptible to inactivation by subsequent mutations

The ultimate success of proviral targeting is critically dependent upon improved and efficient delivery systems

Dr Dos Santos (Rio de Janeiro) describes the effects of the combination of Ingenol-B and cART to SIV251 infected rhesus monkeys.
Ingenol-B is a HIV reactivating agent that belongs to the family of PKC agonists.

Previous works have shown Ingenol-B is able to reactivate latent HIV-1 in infected primary cells and immortalized cell lines as J-Lat.

It is also more potent than other known reactivators like SAHA, TNF-α, PMA and HMBA

 

The authors developed a study with two females rhesus rectally infected with SIV251 for at least 3 months.

These two animals were submitted to a 9 week treatment of Ingenol-B.

The treatment also followed a on-off week scheme, with Ingenol-B taken on odd weeks, and the animals resting on even weeks.

The animals received two oral doses per day of the drug and blood samples were collected throughout the treatment, and biopsies were collected by the end of the 9 weeks.

They observed both an increase in viral load and in viral diversity in these 2 animals.

 

To test the hypothesis that Ing-B can also reactivate latent virus in a context of cART and viral suppression they have designed a new trial.

In this study they used 6 female rhesus separated in two groups.

Both groups were rectally infected with SIV251 for 8 weeks, then cART was given for 24 weeks for both groups.

The tested group also received two cycles of Ing B for 4 weeks in addition to cART.

And finally both groups were submitted to a washout phase where all the drugs were removed.

Blood samples were taken weekly for each subject animal throughout the trial and viral loads, CD4 and CD8 counts and biochemistry and behavioral parameters were measured.

 

There were no major difference between the treated group and the control group, with the exception of one monkey, the S3 that managed to control the viremia below 10 to the 3rd after the rebound on the washout phase.

They applied the same methodology used in the first trial to measure virus diversity and we we saw no significant difference in virus diversity before ART and during washout for each monkey.

They then depleted the CD8+ t-cells from the S3 animal and kept monitoring the viral load.

What they saw following this procedure was a major increase in viral load reaching levels of 10 to the 7th, that went down few weeks after as the CD8 cells went up.

This show that CD8 response had a major role in controlling the viremia in this monkey.

 

Dr Mailliard (Pittsburgh) studied the potential of dendritic cells to both induce the "kick" and the "kill" phases.

DC1 are programmed to express desired traits to drive cell mediated immunity

DC1 (IL-1b, TNFa, IFNa, IFNg, PolyIC) (Mailliard et al, 2004, 2013; Zaccard et al, 2015)

 

In summary for the "kick phase":

 

•DC1 are superior inducers of HIV latency reversal (mechanism?)
•DC1 “kick” more pronounced with presence of antigen

 

In summary for the "kill" phase:

 

•DC1 prime naïve CD8+ T cells to become effective CTL killers against HIV-1 reservoir Ag during cART.
•Targeting conserved as well as escape variants
•High degree of dysfunctional memory CTL responses to HIV-1 reservoir Ag during chronic infection.
•Cytokine production in absence of killing
Dr Coiras (Madrid) underlined the fact that tyrosine kinase p56 LCK is essential for the activation of T cells.
Dasatinib:
-is a tyrosine kinase inhibitor
-interferes with SAMHD1 phosphorylation, avoiding its deactivation,
-interferes with HIV-1 retrotranscription and proviral integration in vitro,
-is not a viral fusion inhibitor.
She consequently obtained cells from 5 patients treated with Dasatinib and these cells showed very low HIV-1 integration after infection ex vivo.
In conclusion:

•Dasatinib is an Src/p56Lck inhibitor currently used in clinic for treating CML that interferes with HIV-1 replication at two levels:
•Interfering with T-cell activation and proliferation.
•Preserving the antiviral function of the innate factor SAMHD1.
•CD4+ T cells from patients on chronic treatment with Dasatinib were resistant to SAMHD1 phosphorylation upon activation with PHA/IL-2.
•CD4+ T cells from CML patients were refractory to HIV-1 retrotranscription and, consequently, to proviral integration.
•Preserving SAMHD1 antiviral function with TKIs such as Dasatinib could be helpful to reduce the reservoir size during acute infection, creating a more favorable scenario for future interventions aimed at HIV-1 cure.
Dr Henrich (Boston) studied 15 patients with HIV and malignancies, mainly lymphomas who received chemotherapy.
He observed, in some patients, an increase in HIV-1 DNA following chemotherapy and hypotezised that it could be due to reactivation of herpes viruses, as most HIV-1 DNA was isolated from EBV or CMV specific CD4+ T cells.

Dr Sung (Chapell Hill) showed that it is possible to use DARTs to redirect cytotoxic T cells to recognize and kill relevant and diverse virus reservoirs.
 Clearance indicates relevant and sufficient viral antigen originating from replication-competent virus is expressed following Vorinostat exposure
DARTs may be powerful tools in combination with vorinostat to deplete the latent reservoir.

Finally, Dr Abdel Mohsen (San Francisco) proposed that Galactin-9 be further studied as it is able in vitro to produce reactivation of latent HIV.

CLOSING SESSION: you can download by clicking on the link below the Take Home Messages of the workshop presented by David Margolis (Chapel Hill).

 


Download the Take Home Messages


 



 
Key words: HIV cure, HIV eradication, HIV persistence, HIV reservoirs, HIV workshop
Last Updated on Tuesday, 15 December 2015 17:04
 

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