Community Research and Development Information Service - CORDIS

FP7

CUT'HIVAC Report Summary

Project ID: 241904
Funded under: FP7-HEALTH
Country: France

Final Report Summary - CUT'HIVAC (Cutaneous and Mucosal HIV Vaccination)

Executive Summary:
The devastating consequences of HIV disease for both individuals and society make the development of safe, effective and easily administrated vaccines a critical priority in the overall plan to contain the global AIDS epidemic. New approaches addressing methods of cutaneous vaccination with the benefits of a needle-free method have then brought novel insight in immune responses to vaccines. Among those, targeting of professional antigen-presenting cells (APCs) of the skin surface is thus fundamental for rational vaccine design because they initiate, maintain, regulate and orientate adaptive immune responses. In this frame, the CUT’HIVAC cutting-edge approaches are based on vaccine design as well as an innovative needle-free route combined to conventional route of vaccination against HIV in a perspective of human clinical trials where new vaccine candidates will redirect immune response.
The objectives of the CUT’HIVAC project is to develop preclinical and clinical approaches to design new immunogens and vaccine delivery systems that can shape immune response towards the control of HIV disease. Candidate HIV vaccines will be tested for mucosal and transcutaneous application on (i) human skin and mucosal explants, (ii) humanised-mice and animal models and finally (iii) human beings for prophylactic as well as therapeutic approaches against HIV disease.

Project Context and Objectives:
The Human Immunodeficience Virus (HIV) is one of the greatest health challenges the world faces from nearly as long as 35 years. Despite significant efforts over the past decades to design and implement new vaccines strategies against HIV (including inactivated-live virus, peptides, proteins or non-replicative vectors), no truly effective HIV vaccine is yet at hand. Many problems related to specific properties and the diversity of the virus as well as the definition of immune correlate remain to be overcome. The development of safe, effective and easily administrated vaccines is however a critical priority in the overall plan to contain the global AIDS epidemic. Antiretroviral therapies, while available, are not curative and prohibitively expensive for most people in developing countries, and most specifically in Sub-Saharan Africa. As infectious disease endemic areas suffer from poor accessibility to drugs and sanitary conditions, a needle-free method would benefit all infected individuals irrespective their origins and living conditions compared to conventional immunization techniques (i.e. intramuscular or intradermal injection, with their ensuing downsides and hazards like unsterile equipment or vaccine reconstitution, instability of the vaccine preparation, injury). New approaches addressing methods of cutaneous and mucosal vaccination has also brought novel insight in immune responses to vaccines.

Among those, targeting of antigen-presenting cells (APCs) of the skin and mucosal surface are fundamental for rational vaccine design because they initiate, maintain, regulate and orientate adaptive immune responses.

The postulate is that an HIV vaccine should enhance both broadly cross-reactive mucosal humoral and cytotoxic cellular immune responses specific to HIV antigens. While particularly cytotoxic effector T-cell arms is needed to control viral dissemination in both prophylactic and therapeutic approaches, broadly reactive anti-HIV neutralizing antibodies at the site of viral entry e.g. mucosal tissues has become today the most prominent priority in the HIV-vaccine field. Hence, effective prophylactic vaccination strategies should be able to elicit mucosa-associated immune responses to prevent spreading of the virus at the site of natural primary infection whereas therapeutic vaccination should also elicit strong and efficient CD8 responses.

CUT’HIVAC project aims at assessing a new HIV vaccine strategy to prevent and control HIV infection based on transcutaneous and/or mucosal needle-free vaccination.
CUT’HIVAC will combine transcutaneous and/or mucosal routes and immunogen delivery systems to preferentially promote and redirect immune responses towards high level of mucosal Abs and effector CD8 cell responses directed against various HIV antigens. Taking advantage of new genetic and immunological information provided by scientific, industrial and academic partners, CUT’HIVAC will design, develop and validate innovative immunogens and delivery systems as well as immunization methods. Both clinical trials and preclinical models of immunogenicity of HIV vaccine candidates by transcutaneous and mucosal routes will be implemented. It also aims at rapidly translating preclinical approaches into prophylactic and therapeutic clinical trials in developed and developing countries. Therefore, CUT’HIVAC will use emerging, highly cutting-edge knowledge and technologies and capitalize on recent discoveries in genetics, immunology as well as vaccinology to design innovative therapeutic and prophylactic vaccine strategies against worldwide HIV infection with a view to widen its application to other infectious diseases such as malaria and tuberculosis.

Confronted to the complexity of immunological events related to HIV infection, it can be expected that only combinations of multiple new approaches will succeed in controlling and eradicating it. Promising approaches for innovation include (1) modifications of the modality of vaccine applications (2) better targeting of a high number of antigen-presenting cells (APC) (3) a mechanism improving APC activation and vaccine compounds-uptake (4) new vaccine candidates for dealing with viral diversity and (5) new therapeutic and prophylactic approaches. The cumulative expertise of CUT’HIVAC partners are organized around these concepts in a perspective to propose an answer to control HIV disease.

The objectives of the CUT’HIVAC project is to develop preclinical and clinical approaches to design new immunogens and vaccine delivery systems that can shape immune response towards the control of HIV disease. Candidate HIV vaccines will be tested for mucosal and transcutaneous application on (i) human skin and mucosal explants, (ii) humanised-mice and animal models and finally (iii) human beings for prophylactic as well as therapeutic approaches against HIV disease.

The scientific objectives of the project are to:
• evaluate novel design of HIV genetic vaccines for developed and developing countries,
• evaluate safety and immunogenicity of transcutaneous and/or intravaginal (IVag) applied HIV vaccines in prophylactic vaccination,
• develop a new therapeutic approach combining drugs and HIV vaccine by transcutaneous delivery in HIV-infected individuals to control HIV,
• understand skin and mucosal mode of administration for the development of new needle-free vaccination strategies against HIV in:
- in vitro preclinical models including skin and mucosal human explants,
- in vivo preclinical models of engrafted skin and T-cells humanised mice,
• understand the molecular basis of immune HIV pathologies,
• redirect immune responses toward mucosal and cytotoxic T-cells,
• develop capacity building and inter-laboratory standardisation between developed and developing countries.

The technological objectives of the project are to:
• develop the transcutaneous and intravaginal methods of HIV application in humans,
• optimise standard operating procedures of needle-free method of skin administration operating procedures of vaccines,
• optimise standard operating procedures of needle-free method of intravaginal mucosal administration of vaccines,
• advance existing standardisation in immuno-monitoring,
• develop new genetic HIV vaccine,
• develop novel adjuvants,
• develop and validate humanised-mice model (Human skin explant-engrafted with peripheral T-cell reconstitution).

Project Results:
WP1 – Design and development of innovative HIV vaccine candidates
Main objectives
- To design and develop new vaccine candidates addressing the extreme diversity of HIV genes
- To combine and optimise cutting-edge tools and technologies to develop efficient delivery system
Main results
- on vaccine design:
UREG established a high yield production workflow for HIV Gag-VLP production and purification. Fluorescent VLPs are generated by staining with carboxyfluorescein succinimidyl ester (CFDA). As proof-of-principle, uptake of labelled VLPs into myeloid derived DCs has been proven. HIV derived VLPs of Gag-GFP-fusions were inferior. Material was provided to partner P5-Charite as well as UPMC-A and -D for analysis of uptake into skin cells (WP3).

Similarly, for the study of the uptake and processing of VLPs in the skin, UPMC-C designed fluorescent MLV VLPs. The fluorescent particles were made with MLV-Gag capsid proteins fused with the green fluorescent protein (GFP) and pseudotyped the GP140 HIV envelope protein derived from HIV-1 clade B GP160 envelope protein (JRFL strain). Fluorescent MLV VLPs were produced in UPMC-C and sent to UPMC-A team for the study of penetration pathways in mice and to CHARITE group (P5) for the study of penetration and antigen presenting cells activation in human skin explants. MLV-Gag VLPs pseudotyped or not with HIV envelope will also be prepared and sent to UPMC-D for study the antigen processing in human dendritic cells. Control VLPs (non-pseudotyped) have been also produced and send to the partners.

CNRS has designed all along the program a large range of fluorescent poly(lactic acid) (PLA) nanoparticles in order to evaluate the uptake efficiency of such vaccine vehicle by different antigen presenting cells, either in vitro or in vivo. By developing a simple process of production, we have been able to incorporate in the core of PLA nanoparticles during the nanoprecipitation process any kind of hydrophobic fluorophores. Such properties have allowed the tracking of vaccine candidate in skin after topical administration or mucosa, as exemplified by the numerous papers describing such finding, with the groups of B. Combadière (UPMC-A) and A. Vogt (Charité).

FIT Biotech provided GTU -EGFPluc which expresses the enhanced green fluorescent protein-luciferase fusion protein (EGFPluc) to several partners to be further evaluated in in-vivo expression studies (UPMC-A and Charité). UPMC-A has also validated DNA-GTU® expression by fluorescence and bioluminescence, we carried out a transfection assay in COS-7 monkey cell line with in vitro JetPEI. One day after transfection, fluorescence and bioluminescence were evaluated in IVIS® Spectrum Imaging System

- on immunogene design
Another objective was to design optimized immunogens capable of eliciting broad cross-clade immune responses to tackle the problem of HIV diversity. For this, UREG has developed Gag and Pol variants enriched with T cell epitopes from all clades were generated. UREGs universal epitope-enriched Gag variants that qualify as next-generation antigens were successfully designed. Qualitatively they resemble the wildtype Gag protein concerning functional (i.e. driving formation of VLPs) properties, while at the same time resembling broad, cross-clade immunogens. Pan Gag Constructs and derivatives thereof are available for the H2020 funded program EHVA (European HIV Vaccine Alliance) as DNA vaccines, for delivery by various viral and non-viral delivery systems as well as recombinant VLPs.

Irsicaixa and IMPACTA have tested more than 1000 individuals with existing HIV infection in collaboration with centers in Boston and South Africa and, based on the data generated in Barcelona and Lima, designed a novel HIV T cell immunogen sequence referred to as ‘HTI‘ (HIVACAT Immunogen sequence). This sequence covers the targets of T cell responses that the Lima studies have found to be associated with reduced viral loads in untreated individuals and contains more than 50 optimally defined CTL epitopes that can be targeted in the context of at least 70 different HLA class I molecules. The HTI sequence is overall enriched for conserved segments of the viral proteome and contains most described hotspots of CD4 T cell responses as well
The entire HTI sequence has been cloned by now into DNA, MVA, Chimpanzee adenoviral vector and BCG-derived vectors and a lentiviral construct, all of which will be further developed in the new EAVI 2020 consortium.

For the generation of improved Env immunogens regarding enhanced stability and affinity to broadly neutralizing antibodies (bNAbs), as well as reduced affinity to non-neutralizing antibodies, UREG developed new screening strategies based on different membrane-associated trimeric Env libraries. UREG is now able to select Env variants with optimized binding profiles to bNAbs and non-neutralizing antibodies using two different screening approaches and different Env libraries. The immunogens generated with these approaches will now be tested in small animals for altered immunogenicity profiles.

Based on its previous experience, UREG provided PAN HIV Gag- VLPs spiked with HIV-1 Env’s derived from a clade C envelope (ZM96 and, alternatively, CN54), which were stably anchored on the VLP surface via the original HIV-Env transmembrane domain (gp145). VLPs could be produced at medium scale following co-transfection of HEH-293S cells and purified from cell culture supernatants by sucrose density centrifugation. VLPs were characterized biophysically by means of determining their Svedberg coefficients and biochemically by assessing their antigenic profile. Alternatively, derivatives of gp41 designed towards exposing the MPER region, respectively, were anchored on the VLP surface and characterized as above. Selected VLP preparations were tested regarding their immunogenicity in small animals (mice, rabbits) either alone, or subsequent to priming with DNA vaccines expressing homologous Gag and Env constructs. Antibody responses were moderately neutralizing some selected Tier 1 isolates.

UPMC-C has developed an alternative vaccine platform based from MLV virus-like particles (VLPs). These VLPs offer the unique advantage to display heterologous antigens, including HIV envelope glycoproteins, in their native conformation that is a pre-requisite for optimal neutralizing antibody induction. In CUTHIVAC project, we designed epitope-enriched MLV VLPs and validated that such particles can be used to induce Env antigen-specific memory CD8+ T cells. Validation was done first with model antigen inserted into Gag (cf. M12 report) and then with HIV-derived polypeptides (M24 report). As a result, MLV VLPs were included in the vaccine delivery studies (WP1) and the preclinical studies with the specific aim to induce HIV-specific mucosal Env specific immunity (WP4).

Partner CNRS exploited the know-how gained with the encapsulation process of hydrophilic molecules (see above: fluorophores) into the core of PLA nanoparticles to incorporate different immunomodulatory molecules involved in innate immunity. Indeed, such incorporation of immune molecules permits to amplify their adjuvant capabilities through PLA particle delivery. The most striking results is the observation that PLA particles loaded with HIV vaccine antigens and Nod ligand could induce strong mucosal immune response either in vaginal or rectal compartments after sub cutaneous administration, such effect being as strong as using cholera toxin and mucosal vaccination (Pavot et al, 2016). This result illustrates the potency of the PLA nanoparticle platform for directing mucosal immunity, when using innate molecules as adjuvants.

Selected HTI derived Gag and Pol sequences of 15-20 amino acids in length have been synthesized by Irsicaixa as highly purified synthetic peptides and coupled to recombinant HSP proteins (HSP70 and HSP90). Mass-spectrography was conducted to monitor HSP loading and the constructs used for subsequent immunogenicity analyses in mice. Overall, even stoichiometric loading of the different peptides was observed, largely irrespective of their length and charge. However, inferior immunogenicity was observed when using HSP constructs compared to DNA delivery, supporting our decision not to move the HSP to a clinical trial setting.

In addition to generating PAN HIV Gag-VLPs presenting the complete external part of the Env trimer on the VLP surface, UREG engineered short sequences from Env's membrane-proximal external region into Cholera-toxin B (CTB) which serves as a scaffold to present such Env-mimotopes. Via a transmembrane-domain the CTB-Env was anchored into Gag-VLPs where it formed pentamers, as expected, so that the CTB component can function as an intrinsic adjuvant by binding to the GM1 ganglioside-receptor on target cells. Presence of CTB enhanced Gag-specific systemic and mucosal humoral immune responses. Mimotope-specific antibodies were induced in a gp41-DNA-prime, VLP-CTB-mimotope boost regimen in mice and rabbits, eliciting moderately neutralizing activity.

Potential impacts:
- Scientific breakthrough:
The group of partners has successfully developed and validated a broader panel of vaccine including virus-like particle based delivery systems (HIV-Gag, MLV-Gag, PLA), which allowed a careful analysis of skin-mediated penetration and uptake of VLP based immunogens. Further to that, algorithms have been devised based on publically available and within this network generated T cell data, which enabled the design of novel T cell immunogens with the potential to induce broader HIV specific T cell responses (CD4 and CD4) towards ideally conserved target epitopes. Furthermore, HIV derived Env with immune-stimulatory components either incorporated into or presented via recombinant or PLA VLPs can induce strong mucosal immunity by a subcutaneous route.

- Economic impact (health costs, market,...):
The know-how gained through WP1 concerning the encapsulation of hydrophobic molecules has been a key element leading to the creation of a start-up, Adjuvatis (www.adjuvatis.com). Furthermore, knowledge gained and IP filed regarding the generation of next generation VLP based immunogens may in the future directly or indirectly translate into vaccine-related products, the latter not necessary being limited in their use to HIV related applications.

- Societal impact (quality of life, health, education, employment, citizen awareness,...):
The developed VLP based immunogens will contribute and did already translate into novel vaccine concepts, which will be further pursued in the 2 H2020 programs EHVA and EAVI. The availability of an even only partial protective HIV vaccine will certainly help to limit the infection in particular in developing countries and thus contribute towards improving quality of life, health, employment and with that also economic strength of these countries.

- European competitiveness :
Key partners of this network are now also engaged in the 2 H2020 HIV vaccine programs EHVA and EAVI, both at least in part building on the achievements of CUTHIVAC. Said this, and regarding the scientific strength of the 2 recently awarded programs, CUTHIVAC clearly contributed towards increasing European competitiveness in the HIV field and also beyond.

WP2 – GMP production & PLA nanoparticle platform
Main objectives
- To provide a delivery system platform of HIV vaccine candidates based on biodegradable nanoparticle strategy
- To supply GMP batches of first generation HIV Vaccine for clinical trials

Main results DNA_GTU GMP production for clinical trials
Production of the drug substance (bulk purified plasmid DNA) and final investigational product is carried out in GMP environment, in the clean room area of the FIT Biotech Ltd.
The manufacturing processes performed in FIT’s facility fulfil the current Good Manufacturing Practices (cGMP) requirements and provide plasmid DNA preparations suitable for use in clinical phase I / II studies. The GMP production batches FIT1103 FIT1303 were manufactured and released for use in the preventive and therapeutic trial (EudraCT no.2011-003171-11 and EUDRACT 2013-004023-37).

The CNRS partner has focused its effort on the design and the elaboration of sterile and non-toxic PLA nanoparticles taking into account our previous expertise and some collaborative work with private industry. We optimized the nanoprecipitation process, through careful monitoring of each step, reducing the amount of residual solvent, ethanol and acetone. We could prepare on a reproducible manner and upon request any size of particles, from 120 nm to 450 nm of diameter, with 2 year stability at room temperature, with no loss in colloidal stability. We have also designed an integrated process, according to EU pharmacopoeia requirement (less than 5000ppm of residual solvent per batch), and a GLP process with all quality control, ensuring an easy translatable manufacturing process for GMP requirements. Indeed such robust process has been a key element towards the decision of creating a new start up, built on this know-how, to ensure exploitation of results gained through this project. See the web site of Adjuvatis, (www.adjuvatis.com), the company created in December 2013, which commercializes the PLA particles. Furthermore, an innovative sterilization process based on irradiation has been elaborated without modifying colloidal properties of these particles, which is a strong added value for clinical purpose.

Lastly, during the elaboration of the GLP production process of PLA particles, we have developed the synthesis of the PLA polymer, using the technology developed by the company Phusis. By mastering the synthesis of the polymer (different Molecular Weight could be prepared), we could modify key parameters for favoring biodegradation. We take advantage of such expertise to design a new vaccine vehicle using micelles and nanoprecipitation process of a specific co-polymer, based on PLA polymer backbone.

IrsiCaixa has evaluated different providers for the production of GMP-based DNA and MVA-vaccine products expressing HTI and has selected, based on competence and price, University of Bristol as the final provider for the DNA production and IDT in Germany for the production of a GMP grade MVA vaccine product. The manufacturing of these vaccine products has advanced well, with both of them being currently at the stage of clinical batch manufacturing. Toxicology studies have been discussed with the regulatory agency in Spain and are ongoing as well. A clinical trial I plan has also been presented to the agency and received positive feedback. A first in human phase I safety trial in HIV uninfected subjects is planned for the end of 2016.

*Potential impacts
Scientific breakthrough:
- Production of bulk purified plasmid DNA and final investigational product in GMP environment suitable for use in clinical trials
- We have been able to prepare a new vaccine (or drug) delivery system based on micelles, using a nanoprecipitation process and PLA polymer as backbone. Such micelles could permit either the delivery of huge amount of peptide/proteins (ref. 38) or specific immune-stimulatory molecules (Jiménez-Sánchez G et al, 2015 Pharm Res.)
- More recently, we have also demonstrated that PLA nanoparticles could be incorporated into a scaffold of nanofibers, which open a new field of research in tissue engineering, for designing “intelligent scaffold”, where nanoparticles permit to attract specific cells for colonization of a scaffold (ref. 67)

Economic impact (health costs, market,...):
- A start-up has been created in December 2013 to develop the know-how gained through the cuthivac program (www.adjuvatis.com). This company is specialized in the development, manufacturing and formulation of biodegradable particles, called i-Particles®, and this innovative technology has multiple application, such as: 1) generation of new antibodies (polyclonal and monoclonal) using hard to use antigens formulated on these particles.2) development of new adjuvants for safer and more efficient vaccines (animal & human vaccines) 3) offering new protein delivery solutions

WP3 – HIV vaccines penetration into human skin and mucosal layers
Main objectives
- To optimise the standard operating procedure of transcutaneous and transmucosal vaccine delivery.
- To determine the efficacy of the penetration of HIV vaccine delivery system into human skin and mucosal layers

Main results
Skin explant model for vaccine penetration
In order to investigate skin penetration of candidate vaccines in cultured human skin, we complemented our previously established incubation in a humidified chamber with a novel protocol for ex vivo tissue culture using cell culture inserts with mesh sizes which require active migration of dendritic cells to enter the tissue culture media. The model enables investigations on freshly excised human skin obtained by patients undergoing plastic surgery for up to 40 hours. Penetration, cellular uptake in cells from epidermis, dermis as cell culture media could be investigated as well as cytokine expression in tissue and release of cytokines in the culture media. The combination of the tissue culture models and cell analyses –as shown in the figure 1 below- enabled us to provide comprehensive preclinical data on penetration, cellular uptake, immune cell activation as well as the cytokine milieu in the skin in response to vaccine administration.

Penetration and cellular uptake of candidate vaccines in ex vivo cultured human skin:
As described in the work plan, candidate vaccines were provided by the CUTHIVAC partners FIT-Biotech, CNRS, UPMC-C and UREG. Results for each vaccine’s architecture obtained by using the above mentioned experimental models are described in the attachment.

In summary, various vaccines produced in WP1/2 have been tested on human skin explants in these developed assays:
- Penetration through various skin pathways (i.e. epidermal, dermal...)
- Study of vaccine up-take and activation of antigen-presenting cells
- processing of antigens by antigen-presenting cells
- in vitro activation of HIV-specific T cells
- induction of inflammatory chemokines and cytokines
These results have direct impact of the choice of delivery vectors for skin immunization for optimal induction of adaptive immunity.
MVA viral vector vaccine recombinant for HIV is suitable vaccine candidate for transcutaneous application or intradermal delivery.


In vivo Studies: Exploratory non-drug clinical trial on the effects of Cyanoacrylate Skin Surface Stripping on skin barrier function
Translation is the core subject of the CUTHIVAC project. In order to further assess the safety, invasiveness and acceptability of CSSS-based transcutaneous vaccination in vivo, we performed an investigator-initiated exploratory clinical trial « Effects of Cyanoacrylate Skin Surface Stripping on Skin Barrier Function CyTech » (Principal Investigators A. Vogt, U. Blume-Peytavi, ethics approval EA1/132/13) in the Clinical Research Center for Hair and Skin Science at Charite. The study also aimed at assessing the optimal time windows for vaccine application after skin pre-treatment as well as possible pro-inflammatory effects of the transcutaneous vaccination method in skin. The exploratory clinical non-drug study was conducted in 12 volunteers. CSSSS-based skin surface stripping was compared to regular adhesive tape stripping by means of skin physiological measurements as well as collection of skin surface material and analysis for inflammatory cytokines. Acceptability and pain perceived during vaccinations were assessed using questionnaires handed out to the volunteers and visual analogues scales for pain assessment. We found that CSSS was well tolerated and well accepted by all volunteers. We found no indication for marked inflammation within the first 48 hrs after the procedure. Skin barrier disruption appeared to be only slightly higher compared to regular adhesive tape. However, it needs to be noted that our experimental ex vivo studies showed that CSSS significantly increase follicular penetration and helps create depots for vaccine which cannot be removed by conventional adhesive tape. Thus, the in vivo data are very reassuring with regard to safety, but the true surplus value of CSSS is probably not the more vigorous barrier disruption, but also the increase of the vaccine reservoir. The clinical data obtained in the CyTech study further suggest, that there is an optimal time window for vaccine application directly after removal of the tape, i.e., both steps should be performed shortly after each other.

Taken together, the ex vivo and in vivo studies conducted within T3.3. of WP3 yielded relevant preclinical data. They were used to prepare safety dossiers for this novel method of vaccine application, which were required to support the ethic committee applications for the CUTHIVAC trials in Lima and London.

*Potential impacts
- Scientific breakthrough:
The observation, that effective uptake of particulate vaccines like VLP occurs by cutaneous antigen-presenting cells when applied after minimal-invasive physical skin pre-treatment procedures like CSSS received considerable attention the field of skin barrier research and dermato-pharmacy, e.g. the work on VLP penetration received a poster award at the annual meeting of the German Society of Dermato-pharmacy (see dissemination). The general concept, that molecules >500 Daltons are not capable of penetrating human skin is essentially challenged by our work. Such penetration clearly does not occur in large quantities, yet immune-stimulatory signals provided by skin pre-treatment could effectively help to target the skin immune system. Vaccine integrity during skin penetration is key. As a result, VLPs are highly interesting candidate architectures. Nanoparticle-based formulation could even increase skin barrier penetration, but have to be constructed in a way that the load is retained until all cargo has reach the target cells.

- Economic impact (health costs, market,...):
Vaccine delivery systems can be evaluated by strict minimal biological assessment with direct impact of the choice of delivery vectors for skin immunization for optimal immunization reducing the use of animal for in vivo testing.
These results have also allowed creating a “technical platform” for testing compounds on live skin tissues: SKINSE (Skin systems evaluation) has been developed by UPMC-A and could be translated into start-up.

- Societal impact (quality of life, health, education, employment, citizen awareness,...)
CUT’HIVAC has developed technologies, generated from advanced knowledge in life sciences and biotechnology that has been largely spread and translated into new therapies, vaccines and clinical practice. Potential entrepreneurial spin-off has and might continue to emerge as a result of the collaboration in the project would obviously be highly competitive and thus would participate in increasing European competitiveness in the biotechnology sector.

- European competitiveness :
CUT’HIVAC has strongly participated not only in fostering European research excellence but furthermore knowledge-based entrepreneurship. UPMC is now engaged in the H2020 HIV vaccine programs EAVI.


WP4 – Preclinical studies of immunogenicity and efficacy of vaccine candidates in experimental animal models
Main objectives
- To identify the best HIV candidate vaccines and routes of immunization that can redirect immune responses toward mucosal and CD8 cell responses for future clinical trials.
- To increase knowledge of the impact of skin and mucosal penetration of vaccine compounds and the quality of immune response.

Main results Immunogenicity of vaccine in murine models
Humoral and cellular immunity is required for protection against invading pathogens such as HIV. We investigated both aspect in murine models and define early in vivo mechanism of immunogenicity of various vaccines:
UPMC-C has developed a vaccine platform derived from MLV virus-like particles (VLPs). These VLPs offer the unique advantage to display heterologous antigens, including HIV envelope glycoproteins, in their native conformation that is a pre-requisite for optimal neutralizing antibody induction. In CUTHIVAC project, we designed epitope-enriched MLV VLPs and validated that such particles can be used to induce antigen-specific memory CD8+ T cells. Validation was done first with model antigen inserted into Gag (cf. M12 report) and then with HIV-derived polypeptides (M24 report).
As a result, MLV VLPs were included in the vaccine delivery studies (WP1) and the preclinical studies with the specific aim to induce HIV-specific mucosal immunity (WP4).
For the study of the uptake and processing of VLPs in the skin, we designed fluorescent MLV VLPs. The fluorescent particles were made with MLV-Gag capsid proteins fused with the green fluorescent protein (GFP) and pseudotyped the GP140 HIV envelope protein derived from HIV-1 clade B GP160 envelope protein (JRFL strain). Fluorescent MLV VLPs were produced in UPMC-C and sent to UPMC-A team for the study of penetration pathways in mice and to CHARITE group for the study of penetration and antigen presenting cells activation in human skin explants. MLV-Gag VLPs pseudotyped or not with HIV envelope will also be prepared and sent to UPMC-D for study the antigen processing in human dendritic cells. Control VLPs (non-pseudotyped) have been also produced and send to the partners (cf. reports M36, M48, M60 and M72).

UPMC-A dissected mechanism of skin cells in the induction of cellular and humoral responses. We used conventional mice as well as KO and Tg models of genes involved in cell migration and immune responses (TCR tg, KO chemokine receptor, Langerin-DTR-egfp Tg mice) for the study of bio-distribution of vaccine in skin (site of immunization by transcutaneous or intradermal routes) and draining lymph nodes (site of immune responses). Bio-distribution of vaccine compounds (CNRS : HIV-P24nanoparticles, UPMC-C: VLP) in the draining LN as well as phenotypically distinct APCs will impact on the quality of the immune responses. We found : 1) the importance of inflammatory cells at the site of immunization in the induction of memory cells, 2) molecular inflammatory signature by transcriptomic analysis for the induction of T follicular helper cells and defined a key molecule produced by skin migratory cells for the induction of T follicular cells (Levin et al. manuscript in preparation; Bonduelle et al. manuscript in preparation).The basic understanding of such mechanism will have a great impact in adapting dose, route and adjuvant usage in vaccination strategies.

As described in WP1, the final HTI T-cell immunogen (Irsicaixa) sequence has been designed at Irsicaixa, codon optimized and incorporated into DNA, MVA, Chad, BCG and Lentiviral vectors and, in form of synthetic peptides, also coupled to HSP. Murine immunogenicity studies have been conducted mostly as DNA and MVA prime/boost strategies while additional combinations using Chad and BCG have only been tested in small number of animals to date. These combinations will be further developed and optimized in EAVI2020 providing seamless continuity to the program developed in CUTHIVAC. Macaques studies planned in EAVI2020 will also build on the data generated using the HTI.DNA and HTI.MVA which showed very strong immunogenicity and the induction of effector–memory T cell responses targeting the entire HTI sequence evenly.

Independently, the HTI sequence has also been produced as a mRNA and has been studied for the representation of specific epitopes in in-vitro antigen processing and representation analyses using some of the tools developed in WP1. With positive data and good immunogenicity in mice, this approach has entered now into clinical phase I trial testing in Barcelona within the FP7 iHIVARNA consortium. The phase I trial is being conducted in HIV infected individuals and consists of a dose escalation of the combined HTI/adjuvant mRNA mix (referred to a ‘Trimix’) in a first-in-human safety study.

On the other hand, and as described in more details in the periodic progress reports over the funding period of CUTHUVAC, the delivery of soluble HSP complexes containing HTI peptides has not yielded any reproducible responses after 2 injections. This approach was expanded to include a HSP-HTI sequence delivered as a DNA vaccine; with poor immunogenicity data in murine studies as well. With the approval of the program officer the HSP approach was abandoned and efforts instead shifted towards the execution of the CUTHIVAC 003 trial in Lima.

Aside from HTI immunogenicity testing, Irsicaixa has also initiated immunizations of mice with the dGag sequence in combination with prime/boost regimens using HTI. While in vitro data show clearly that the current version of the dGag protein can form Env-containing VLP, a DNA based co-vaccination of dGag and Env expressing plasmids did yield poor humoral immune responses. Irsicaixa is not completing immune analyses in vaccinated rabbits with the inclusion of in vitro generated VLP products.

Humanized mice model
Two models of humanized mice has been developed by UPMC-A:
1) In order to study antigen-presenting cells and T cell functions in the skin microenvironment, we need a relevant preclinical model that allows conservation of the whole epidermal and dermal skin layers. Here, we describe a model of mice transplanted with human skin that represents a crucial methodological step for studying skin immune responses and for testing new vaccination and therapeutic strategies. We have developed NOD-Scid IL2rγnull (NSG) mouse model transplanted with human skin to study skin immune cells in vivo behavior and functions within 3-4 weeks post-engraftment. We propose a standard operating procedure where full-thickness 1x1cm2 of human skin samples are engrafted onto the back of NSG mice (Supplementary materials and Figure S1). This humanized NSG mouse model engrafted with human skin provides a long-term survival and functional maintenance of human skin immune system in a well-conserved tissue environment. It allows analyzing in vivo antigen penetration in the tissue, antigen uptake and presentation by LCs and dermal DCs, local immune activation and skin-resident T cell responses. With the long-term preservation of a complete human skin immune system, this model offers the unique opportunity not only to better understand mechanisms of skin innate and adaptive immune response but also to test new compounds and devices for cutaneous routes of vaccination, new therapeutic approaches for viral, bacterial, parasite infections but also skin diseases.
2) An adequate model of human skin immunity for in vivo evaluation is lacking. HIS mice represent an attractive tool to study ontogeny of cellular components of the human immune system in vivo. However, little is known about the skin compartment in these animals. We investigated colonization of the skin of HIS mice by human hematopoietic cells in order to establish a preclinical model to investigate vaccination and immunotherapeutics application via the skin route. We produced HLA-A2 transgenic NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-A2.1)1Enge/SzJ) mice humanized for the immune system (NSG-HLA-A2-HIS) with a single intra-hepatic injection of 0.5x105 to 1.5x105 HLA-A2+ CD34+ human hematopoietic progenitor cells (hHPC) into sublethally irradiated newborn NSG-HLA-A2 mice (< 5 days old). This led to a high level engraftment of a human immune system with long-term maintenance capacity in vivo. We demonstrated here that human hematopoietic cells, mainly human T cells, colonized the skin of HIS mice. Complementation with hAPC at the vaccination site allowed the induction of an immune response. This highlighted the potential of the HIS mice has an innovative preclinical model for prospective analysis of vaccination via skin route and testing of DC therapies against infectious diseases as well as cancer and autoimmune diseases. As such, this will also generate knowledge on understanding the early events following immunotherapeutic administration and support demands for in vivo biomodeling of the interaction of immunotherapeutic with the human immune system.

*Potential impacts
Scientific breakthrough
- HTI containing DNA and MVA prime boost regimens show strong immunogenicity in mice and macaques. These data have prompted the GMP production of these constructs for human clinical trial
- Based on the data generated in this WP, recombinant HSP delivery of antigenic peptides does not appear to be highly immunogenic.
- HIV candidate vaccines and routes of immunization that can redirect immune responses toward mucosal and CD8 cell responses for future clinical trials has been identified.
- We have increased our knowledge of the impact of skin and mucosal penetration of vaccine compounds and the quality of immune response in animal models.
- We propose future strategies to enhance mucosal immunity for HIV vaccine that will be used in future projects
- Our work on humanized mice also generate knowledge on understanding the early events following immunotherapeutic administration and support demands for in vivo biomodeling of the interaction of immunotherapeutic with the human immune system.

Economic impact (health costs, market,...):
Vaccine delivery systems can be evaluated by strict minimal biological assessment with direct impact of the choice of delivery vectors for skin immunization for optimal immunization.


WP5 – Prophylactic clinical trials on safety and immunogenicity of HIV candidate vaccines
Main objectives
- To determine safety and immunogenicity of DNA-GTU® and a MVA-B in Phase I clinical trials for HIV negative healthy adults by transcutaneous route
- To propose an innovative prophylactic approach combining transcutaneous and intravaginal routes for 2nd clinical trials using novel-design 2nd generation HIV vaccines

Main results CUT’HIVAC is conducting prophylactic and therapeutic Phase I clinical trials with HIV multi-clade DNA-GTU® candidate or HIV-clade B recombinant Modified-Vaccinia Ankara (MVA) applied by transcutaneous needle-free routes that aim at targeting vaccine compounds into the open hair follicles and/or combined with intramuscular route. CUTHIVAC-001, CUTHIVAC-002 and CUTHITHER001 data management and monitoring has been performed by UCL and in IMPACTA for CUTHIVAC-003

CUT’HIVAC-001 is using a HIV multi-clade DNA-GTU® strategy applied or injected by differential routes of administration including electroporation and needle-free approaches. Thirty (30) HIV seronegative volunteers have fully completed the trial.
All 30 participants enrolled into CUT’HIVAC:001 preventative trial, have now completed all study visits and follow up. Primary safety and immunogenicity endpoint analysis is completed, while extended exploratory immunogenicity analysis is underway to further examine the T cell response. The clinical study report (CSR) is in preparation for submission to the MHRA.). Safety and Immunogenicity report will be delivered in Q3 2016.

CUT’HIVAC-002 expects to enroll 24 HIV seronegative volunteers before Sept 2015. This Phase I trial will apply a HIV multi-clade DNA-GTU® prime and pCN54-ENV protein boost strategy. The trial has been approved by regulatory and ethical bodies and is in recruitment, it is anticipated that it will be fully enrolled by September with last subject last visit anticipated in March 2017 and study report released Q3 2017.

In addition, full safety data on the HIV envelope DNA and protein were produced by Charles River. A Single Dose Biodistribution Study (No 525574) of CN54ENV DNA delivered with electroporation was performed in rats. The results from this study showed that biodistribution of the plasmid is restricted to the site of administration and that there is no persistence of the plasmid in these tissues beyond 60 days. A good laboratory practice (GLP) rabbit tolerance and toxicity study (CR 525569) has been performed to directly support the CUTHIVAC 002 clinical study, where animals received vaccinations with a 2mg dose of CN54DNA given three times at 3 weekly intervals with electroporation, followed by intradermal or intramuscular boosting with CN54gp140 (100ug). All reactions seen were in accordance with what would be expected. There were no worrying local side effects or indications of systemic toxicity. Analysis of the (presumed) peak antibody responses measured 2 weeks after the last immunisation corroborated what had been seen previously in mouse and rabbit studies. These data were used to support regulatory and ethical filings required for the initiation of the CUTHIVAC002 trial.

CUT’HIVAC-003: Since the HSP-based approaches were abandoned in 2012, the teams at Irsicaixa and IMPACTA were able to plan, initiate and fully execute a clinical phase I trial in Lima that compared the safety and immunogenicity of a MVA-B vaccine candidate delivered either by the TC or the IM route. The team profited thereby from the fact that the MVA-B vaccine candidate was available at Irsicaixa essentially free of costs (aside from additional stability testing, shipping and re-labelling related expenses) and the protocol writing was greatly facilitated by members in the consortium working on CUTHIVAC001/002 trials. Overall, the clinical trial was executed rapidly, despite a number of hurdles: the first draft protocol was prepared in English in July 2013 and final approval was obtained by May 2014. The trial was somewhat delayed due to investigator-initiated amendments that were introduced to collect additional mucosal samples as well as to obtain samples for skin and feces microbiota analyses and samples that allowed for a comprehensive assessment of the innate immune response to vaccination. The last subject was enrolled in July 2015 and the last visit was conducted on November 2015. No serious adverse effects have been reported. Samples have been shipped from Lima to Barcelona and Paris and a last shipment is pending for samples to arrive in London. Immune analyses will include a detailed Elispot-based assessment of the breadth and depth of the immune response and an in vitro viral replication inhibition assay (VIA). For the latter, an investigator from Irsicaixa has spent a few weeks in the lab of Lucy Dorrell in Oxford to establish the VIA in Barcelona. This technique will also facilitate the analyses n the clinical trials planned in EAVI2020. In addition, a biosystems approach to identify innate immune signatures induced shortly after vaccination has been applied to the samples from IMPACTA and has shown stronger signals in the IM arm compared to the TC route, at least at day 1 after immunizations. Indeed, UPMC-A has studied the transcriptomic profile of individuals vaccinated by MVA-B by both routes of immunization. Systems biology approaches are used to understand the impact of innate immunity on adaptive immunity (>8 immune parameters including systemic and mucosal responses). These innate immune markers will be rated to the adaptive immune responses to the MVA-B immunizations, as well as to the data emerging from the microbiota analyses. To date, skin microbiota data have been generated at the abs at Irsicaixa. Feces samples will be included in the upcoming shipment from Lima, which also includes mucosal samples that will be shipped to Imperial in London. Safety and Immunogenicity report will be delivered in Q4 2016.

Potential impacts
Scientific breakthrough
From the beginning of the program, we strengthen our capacity to mount preventative clinical trials. We have extremely successful as demonstrated by the launch of 3 preventive clinical trials due to the continuous and strong efforts of partners despite unexpected delay as well as well-planned contingency plans (see 4 amendments). These clinical trials will bring insight into the impact of route of various vaccine administrations (HIV recombinant DNA, attenuated virus, GP120 HIV protein) in human. We have also developed a strong immunomonitoring platform and have adapted our effort to a modern investigation of vaccine immunogenicity in human. This work has strongly contributed to fostering European research excellence as demonstrated by successful continuation in new H2020 project (EAVI).


WP6 – Therapeutic clinical trials on safety and immunogenicity of HIV candidate vaccines in HIV infected adults
Main objectives:
- To demonstrate that TC approach can shape the immune response to HIV by enhancing the CD8 T-cells against HIV in a Phase I/IIa clinical trials using naked DNA-GTU® multiclade HIV vaccine A, B, C and FGH vaccines
- To propose new innovative approaches of therapeutic immunisation against HIV for future trials to be developed in Europe and in developing countries

Main results:
CUTHIVAC launched one therapeutic clinical trial, namely CUTHIV-THER_001.
Imperial College London submitted applications for ethical, regulatory and host-site approval of the CUTHIV-THER clinical therapeutic trial. Initial comments were received from both regulatory authority (MHRA) and the Ethical review committee and the protocol and associated documentation revised accordingly. Full approval was granted with study site initiation on 16th April 2015. The first participant was enrolled on 30th June 2015. The trial was fully enrolled by 30th November 2015 and is due to be completed by April 2016. Safety and Immunogenicity report will be delivered in Q4 2016

Progress: In total 36 participants were screened to enrol 30 participants over a period of 6 months. There are currently 30 males and 0 females enrolled, age range 21-42 years. To date, 12 participants have fully completed the trial with all participants expected to complete by April 2016. There were a variety of reasons for the screen failures such as abnormal liver function tests, low BMI (these were screened in clinic prior to the establishment of a telephone screen where BMI was asked), and some participants withdrew consent between screening and first visit although they had been deemed eligible.

Similar to WP5, in preparation for immune monitoring activities for CUTHIVAC clinical trials, UPMC, Irsicaixa, ICL have developed HIV-specific specific cellular assays and capacity building in Impacta (Peru) and INS (Mozambique). Given limitations in personnel support at IMPACTA in the last year of the CUTHIVAC funding period, it was however decided to conduct the initial immune analyses at Irsicaixa. Still, the activities have helped to build local capacity and the IMPACTA lab is well prepared to conduct further immune analyses.

Over the last 6 years of CUTHIVAC funding period, Irsicaixa has developed two alternative approaches for immune monitoring. This includes a DTH approach in which small optimally-defined HLA-class I restricted CTL epitopes are injected i.d. and the ensuing delayed type hyper-reactivity reaction is used as a measure of successful induction of a vaccine response. The second approach consists of a ’boosted’ flow cytometry in which multiple cytokines are being detected in the same fluorescence channel of standard flow cytometers. By using this novel, boosted cytokine staining/flow cytometry strategy, it is possible to describe new patterns of T-cell response to vaccination that would have been missed by standard assays. Importantly, this approach also allows detecting broad and strong virus-specific T-cell responses in high exposed uninfected individuals. These responses were characterized by a T-helper type 1 cytokine–like effector profile and produce cytokines that have been associated with potential control of HIV infection, including interleukin 10, interleukin 13, and interleukin 22. This novel approach has the potential to significantly improve the current understanding of HIV-specific T-cell immunity as well as allow for a more-comprehensive assessment of host immune responses to vaccination.

Given the widespread use of the VIA in clinical trial setting, Irsicaixa has developed a test virus that allows to avoid potential confounding effects of replication of autologous virus in standard assays. The virus is a double mutant virus that a) includes a Raltegravir resistance mutation and b) a Nef mutation that avoids downregulation of HLA class I molecules. These mutations were introduced with the intention to suppress replication of autologous virus in the in vitro culture by adding Raltegravir to the assay and to increase the signal of T cell mediated suppression of viral replication by avoiding negative effects of the Nef protein on the recognition of viral infected cells.

In addition, Irsicaixa investigators have worked closely with investigators in Oxford on samples from past HIV vaccine trials (STEP, Pamphili) and were able to show that the in vitro inhibition activity is directly related to the degree to which individuals are focussing their virus-specific T cell immunity on the HTI sequence. Importantly, these past and ongoing activities with Oxford also allow the Irsicaixa team to establish and apply the most advanced VIA approaches to the samples from the CUTHIVAC 003 trial in Lima.

Potential impacts
Scientific breakthrough
A sensitive and reproducible, ‘boosted’ flow cytometry approach that can detect responses in HESN but not in HIV unexposed individuals has been established. Such an assay could provide a valuable tool to measure vaccine induced responses and, possibly even more relevant, help in identifying immune responses that may protect at least some HESN from infection. The latter has the potential to greatly guide preventive HIV vaccine development

From the beginning of the CUTHIVAC project, IrsiCaixa fosters close collaborations with the teams in developing countries, especially in Peru but also in Mozambique. As a result, we have ongoing exchange of investigators from Lima in Barcelona and strong interactions especially on cohort establishment in high risk populations where also Barcelona based groups could profit from the advances in Lima. The rapid writing of the CUTHIVAC003 protocol, its translation to Spanish and the initiation of the clinical trial in Lima were other mutually beneficial activities that contributed to the overall achievements of CUTHIVAC.
The data from the collaboration with Mozambique have also been discussed on several occasions, along with providing input for patient recruitment, and in vitro immune analyses. The data generated suggest that the lab in Mozambique has conducted Elispot assays very proficiently and that the selection of antigen could be considered into a clade C vaccine immunogen.
These activities have further established the collaborations between the Lima site and IrsiCaixa on one site and Paris and Mozambique on the other side and brought these collaborators as reliable partners in the CUTHIVAC program. The continuing scientific interactions between all 4 sites will further promote the expansion of local research agendas and their independence and ability to secure future funding to ensure the sustainability of their programs.

Potential Impact:
Scientific, social and economic rationales for the exploration of CUTHIVAC are strong:
A start-up has been created in December 2013 to develop the know-how gained through the CUTHIVAC program (www.adjuvatis.com). This company is specialized in the development, manufacturing and formulation of biodegradable particles, called i-Particles®, and this innovative technology has multiple application, such as: 1) generation of new antibodies (polyclonal and monoclonal) using hard to use antigens formulated on these particles; 2) development of new adjuvants for safer and more efficient vaccines (animal & human vaccines); 3) offering new protein delivery solutions. In addition, a “technical platform” for testing compounds on live skin tissues: SKINSE (Skin systems evaluation) has been developed by partners and could be translated into start-up.
In addition, CUTHIVAC partners foster close collaborations with teams in developing countries, especially in Peru and Mozambique: exchange of investigators, cohort establishment in high risk populations and capacity building.
Thus, CUT’HIVAC has developed technologies, generated from advanced knowledge in life sciences and biotechnology that has been largely spread and translated into new therapies, vaccines and clinical practice. Potential entrepreneurial spin-off has and might continue to emerge as a result of the collaboration in the project would obviously be highly competitive and thus would participate in increasing European competitiveness in the biotechnology sector. Key partners of this network are now also engaged in the two H2020 HIV vaccine programs EHVA and EAVI2020, both at least in part building on the achievements of CUTHIVAC.

List of Websites:
www.cuthivac.eu

Béhazine COMBADIERE - behazine.combadiere@upmc.fr - Phone :+33140779888
Robin SHATTOCK - r.shattock@imperial.ac.uk

Contact

Lucie SALVAUDON, (European Affairs Officer)
Tel.: +33 1 44277279
Fax: +33 1 44277467
E-mail
Record Number: 187170 / Last updated on: 2016-07-20