Programme 5

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.
  1. 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.
  2. 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.
  3. 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.
  4. 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.