Programme 5: Preclinical development and testing of HIV vaccines, antibodies and microbicides
Chair: Felipe García
Co-Chair: Javier Martínez-Picado
The advent of antiretroviral (ARV) therapy (ART) has significantly reduced the morbidity and mortality of HIV-1 infection. In addition, the number of people receiving ARV therapy continues to increase, and recent evidence from clinical trials has confirmed the powerful impact ARV drugs can have on the AIDS pandemic as part of an effective option for HIV prevention. As a consequence of preventive and therapeutic efforts, the global incidence of HIV infection has stabilized and has begun to decline in many countries. However, despite these successes current ART has a number of limitations. This will imply that if no alternatives are found, many new individuals will be infected and many HIV infected patients will die in the next few years. Development of new preventive vaccines, together with other preventive biological tools (as antibodies and microbicides) are necessary to control the epidemic. In addition, functional cure has become a research priority in the HIV field. Therapeutic vaccines to improve the immune response in HIV infected patients could help to achieve this objective.
In addition, extensive collaborations with other national groups (both members or not of the RIS) and international groups and networks (EHVA, EAVI, HIVACAR, iHIVARNA, Gates Foundation,…) are very important to the achievement of our objectives.
Based on this scenario the HIV vaccine, antibody and microbicide Programme of the RIS focuses on the following objectives.
- To develop biological strategies to prevent or contribute to cure HIV infection. The aim would be to develop HIV immunogens capable of inducing an effective cellular and humoral immune response by different approaches. In addition, we plan to develop new prototype of microbicides. We will use animal models and cellular in vitro assays to test the immunogenicity of the prototypes previously developed and the best vaccine candidates will be included in clinical trials (Programme 3).
- To detect and characterize broadly neutralizing antibodies. Antibodies with the ability to neutralize HIV broadly and potently are rare. In addition, the induction of this type of antibodies have been demonstrated as the most important challenge in the development of an effective preventive vaccine against HIV. In addition, in the last few years some bNAbs have been isolated from HIV infected individuals. These antibodies have become an essential tool to protect and control HIV infection. In fact, different recent or ongoing clinical trials are trying to demonstrate their efficacy. We will follow the study of new bNAbs to be used as a preventive or therapeutic tool and be complementary to the study of new preventive a therapeutic vaccines (WP3).
- To improve our understanding of pathogenesis of the control and protection of HIV infection (in collaboration with programme 4: Immunopathogenesis of HIV infection). Despite all the years of research of HIV immunopathogenesis, no clear data are available to explain which mechanism could collaborate to effectively control and protect from HIV infection. To address this issue we will build a platform to evaluate vaccine induced cellular and humoral immune responses in the clinical trials performed in Programme 3. In addition, it is necessary to find new tools to discover markers of response to predict those patients with higher likelihood of response and to try to induce those immune responses that could be crucial in the control of HIV infection. We plan to study the influence of microbiome and other immunologic and transcriptomic surrogate markers of response to vaccines. This is mandatory to further improve our knowledge of protection and control of HIV infection.
These objectives are based on previous scientific background of RIS members but also in different structures and links with other programmes that have been set up in the last years:
- A centralized BioBank, representing the collaborative effort of all the groups involved in the network.
- A close collaboration with programme 3 and 4. Programme 3 has the WP 3 dedicated to perform clinical trials to test new immunogens, microbicides or proof-of-concept studies to assess the relevance of different markers in HIV infection.
Work Package 1 (WP1): Prototypes of HIV immunogens, vectors and adjuvants
Leader: Mª Ángeles Muñoz
Discovery of prototypes of HIV immunogens, vectors and adjuvants.
- New inserts to be included in the different vectors. We will compare the effectiveness to induce robust and broad T-cell responses of different concepts of selection of HIV-1 epitopes to be expressed or codified by a vaccine to redirect HIV-1 specific T-cell responses against vulnerable targets of the virus associated with viral control vs a more classical consensus insert.
- Vectors. We plan to continue working in some classical approaches as bacterial vectors (BCG and MTBVAC), viral vectors (MVA, chimpadenovirus), DNA or VLP. In addition, we will develop new innovative vectors to be tested as preventive and therapeutic vaccines as could be RNA vectors.
- Adjuvants. Some of these vectors should be coformulated to improve their effectiveness or to target dendritic cells (DC). We plan to work with different nanoparticles and molecules to target dendritic cells in RNA based vaccines.
- Microbicides. Based on second-generation carbosilane dendrimers scaffold built from a silicon atom core.
Work Package 2 (WP2): “In vitro” and small animal models to test prototypes
Leader: Montserrat Plana
- “In vitro” uptake and immunological activity of different vectors. We plan to generate a dendritic cell (DC) “in vitro” model to study antigen presentation, capture, immunophenotype, migration capability, and innate immunity changes induced by immunogens in DCs.
- In vivo efficiency. Small animals (mice and rabbits) will be used for testing immunogenicity and safety of the prototypes developed in objective 1. Humanized and SCID-mice models developed in WP1 will also be used for studying cellular and humoral immune responses induced and immunogen distribution.
- We will perform comparative analysis of B and T cell immune responses triggered by the different vectors in a Balb/c mouse model, in comparison with the old vectors, to define the best-in class vector immunogen.
- These models will help to the establishment of immune parameters (antibodies, T cells, cell phenotypes) in a mouse model and selection of a best-in-class prime/boost protocol of vectors in combination developed by other groups.
- A rabbit animal model will be ussed to study the humoral immune response induced by the VLPs generated in WP1. Purified IgGs from immunized animals will be used to evaluate neutralization breadth and to map the epitopes recognized by the induced antibodies.
- Finally, Human BLT-mice, rats and rabbits models will be used for studying of microbicides against HIV infection at vaginal and rectum level.
Work Package 3 (WP3): Detection and characterization of HIV bNAs
Leader: Eloísa Yuste
- We will identify broadly neutralizing responses in different groups of patients. The neutralization patterns and the epitopes recognized by sera from these patients will be characterized.
- Characterization of neutralizing responses with different panels of recombinant viruses and pseudoviruses in different groups of hiv-1 patients. Neutralizing activity will be assessed by neutralization assays based on HIV-1 Env-recombinant viruses expressing a luciferase reporter. We have a panel of recombinant viruses with different subtypes representing the global HIV-1 diversity. Virus infectivity will be determined 48 hours post-inoculation by measuring the amount of luciferase activity expressed in infected cells. The ability of sera/antibodies to neutralize viral infection will be assessed as the percent inhibition of viral replication at each serum/antibody dilution compared to an antibody negative control. Sera from patients capable of neutralizing at least one virus from three different subtypes with a minimum ID50 of 100 will be selected for further characterization.
- Epitope mapping of sera that show the broadest neutralization breadth bNAb specificities directed against major epitopes will be studied. 1-Fine tune mapping of anti MPER antibodies using gp41 contructs. 2-Analysis of CD4bs antibodies by ELISA using mutated gp120 and a competitive flow cytometry-based functional assay. 3- Detection of glycan-dependent neutralizing antibodies by neutralization assays with mutated viruses
- Study of cell-to-cell neutralization in the sera that show the broadest neutralization breadth
- The ability of sera to protect CD4 T-cells from cell-to-cell transfer, fusion or death when cocultured with HIV producing 293T cells will be determined with a previously described cell-to-cell HIV transfer assay.
- We will isolate and characterize new broadly neutralizing antibodies from patients with a broad and potent humoral immune response to HIV. The population of B cells will be characterized and monoclonal antibodies will be isolated from antigen-specific B cells. In addition, the epitopes recognized by these antibodies will be mapped.
- Isolation of monoclonal antibodies from patients that showed that broadest neutralizing responses. We will use a method based in the selection of B-cells expressing IgGs capable of binding HIV envelope proteins in their native trimeric conformation that are expressed in the cell surface.
- Characterization of neutralizing responses with the virus panels and identification of antibodies with the broadest neutralizing responses (bNAbs). We will select the antibodies capable of neutralizing at least one virus from three different subtypes for further characterization.
- Sequence analysis of the bNAb immunoglobulin genes to determine the level of somatic mutation and the length of the HCDR3 regions.
- Epitope mapping of bNAbs isolated, analysis of the ability of the selected monoclonal antibodies to protect CD4 T-cells from cell-to-cell transfer, fusion or death will be determined, the analysis of the viral diversity in patients with bNAbs isolated.
Work Package 4 (WP4): Platforms for immunological response outreading in animals and humans
Leader: José M. Benito
We will constitute a reference laboratory platform for vaccine trial evaluation. We will offer a catalogue of techniques to evaluate vaccine induced immune responses.
- Cellular immune response (innate and adquired). Detection of pro-inflammatory cytokines, Elispot, Intracellular Staining, Proliferative capacity, flow cytometry panels for studing memory T cells and Virus Inhibition Replication assays will be performed in peripheral blood and mucoses. In addition, we will develop a novel flow cytometric analysis ("boosted flow") where we will combine several markers into the same channel of a flow cytometer and can thereby detect many more effector functions than with conventional approaches. This would provide a novel technology for immune monitoring in vaccine trials where we may not know a priori the effect function profile of vaccine induced responses.
- Humoral immune responses. Determination of systemic IgG or IgA responses against gp120, MPER antibodies and antiCD4bs antibodies by ELISA. We will also study the IgG associated neutralization breath against a panel of recombinant viruses with envelopes from 5 different subtypes. We will also test the ability to recognize (by ELISA and neutralization assays) the viral envelope protein included in the vaccine prototype in plasma from vaccinated individuals. In patients showing a significant humoral immune response we will study the neutralization against autologous viruses using a new patented method based in the usage of recombinant viruses including the autologous envelope gene amplified from patient plasma and the Renilla luciferase reporter gene.
- Methodology standardization. We will coordinate assays among RIS labs and constitute a platform for vaccine trial evaluation. We will use international available standards and exchange reference samples for performing quality controls. In addition we will certify the methodology used by National and/or international accreditations.
- Viral escape in response to vaccines. Viral sequences will be analysed in the pre-vaccination time point sample, from the first sample post- analytical treatment interruption (ATI) with detectable plasma virus and at the end of the ATI. The aim is to determine whether the virus rebounding in vaccinees and control patients differs and whether regions covered by the immunogen are under positive selection pressure (versus non-covered regions).
Work Package 5 (WP5): Microbiome modulation of immunological response to vaccines
Leaders: Roger Paredes and Sergio Serrano
We will investigate immunological and transcriptomic surrogate markers of efficacy in vaccine clinical trials. In addition, we will focus on the role of the microbiome in the responses to vaccines in collaboration with Programme 3.
- To investigate surrogate markers of efficacy. Depending on the design of the clinical trial, virologic and immunologic markers that correlate with viral replication control after stopping antirretroviral therapy or with significative reduction of viral reservoir would be investigated. Moreover, surrogate markers of immunogenicity and induction of cellular memory will be investigated.
- Transcriptomic analysis. We will develop and apply systems medicine approaches to identify molecular signatures induced early after therapeutic HIV vaccination that correlate with vaccine induced immune responses and to validate their potential to accurately predict viral suppression after cART interruption. We will profile mRNA and microRNA expression, and generate full proteome cytotoxic T lymphocyte (CTL) epitope maps as readouts of the immune response. Using a variety of methods including predictive mathematical modeling, gene set analysis, pathway analysis, and deconvolution of cell populations. The mRNA, microRNA and CTL profiles will be integrated with existing laboratory results and clinical readouts to define signatures of protective immunity. Validation of the findings from our integrated analysis will be carried out with samples from the new HIV immunotherapy trials performed in Programme 3. In these clinical trials, a structured cART therapy interruption will be performed to assess the viral efficacy of the vaccines. Analysis of post-vaccination samples and clinical parameters with the integrated approach will allow 1) the evaluation of the predictive value of this analysis for immunotherapy efficacy and 2) the determination of the potential of individual parameters for cART interruption as a personalized medicine approach. Data generated will be shared and made available by deposition in a public database.
- Microbiome analysis. We will produce MiSeq™ 16S rRNA sequence data on fecal microbiomes. To characterize alpha diversity, species richness as well as diversity / evenness measurements will be calculated. To assess genus abundance, operational taxonomic units counts will be collapsed to the bacterial genus level and genus proportions will calculated for each sample. In addition, we will perform the functional profiling of the microbiota, a nutritional assessment and nutrient data analysis and will measure soluble plasma markers of enterocyte damage, microbial translocation and systemic inflammation in the clinical trials performed in Programme 3. We will try to find differences in genus abundance to investigate if taxonomic microbial community composition could be associated with response to vaccines.