Final Report Summary - NGIN (Next generation HIV-1 immunogens inducing broadly reactive neutralising antibodies) Executive summary: The elicitation of broadly neutralising antibodies (NAb) remains the primary and most challenging goal in human immunodeficiency virus (HIV)-1 vaccine development. Although a few anti-HIV-1 monoclonal antibodies with broadly neutralising capability have been isolated from infected individuals, none of the immunisation strategies so far explored has proven effective in inducing similar antibodies. Objective of this application has been the development of a variety of next-generation HIV-1 envelope-based immunogens that in combination with new adjuvant formulations are capable of eliciting high-titer broadly NAb responses. Our strategy was based on one side on the identification and cloning of envelopes of viruses that have successfully elicited broad NAbs (bNAbs) in their natural hosts. More than 1 800 HIV envelope genes from primary viruses obtained early after infection of patients, who had successfully mounted bNAbs, were cloned and sequenced. A novel machine learning method was used for understanding the relationship between env sequence and the presence of bNAb. On the other side, we have introduce rational modifications into these and promising HIV-env based immunogens that were already under development by NGIN partners, with the aim of exposing cryptic conserved neutralisation epitopes and permitting their efficient presentation to the immune system. HIV-1 envelopes were expressed in viral vectors or as trimeric (gp140) soluble proteins and screened for their immunogenicity and antigenicity in rabbits. Novel adjuvant formulations were evaluated such as CCL28, the flagellin plasmid (pFlic) and DDA / TDB (CAF01). A selection of envelopes with highest antigenicity was expressed as trimeric envelope-complexes on the surface of virus-like particles (VLP) or virosomes, to further improve immunogenicity. Selected new immunogens were evaluated in prime-boost regimens in rabbits using effective adjuvant formulations. Immunogen / adjuvant combinations that proved most effective in eliciting broadly NAbs were evaluated in non-human primates for their immunogenicity. Project context and objectives: The elicitation of broadly NAbs remains one of the most challenging goals in HIV-1 vaccine development. The preventive action of all commercially available vaccines against viral pathogens today is believed to be mediated primarily through antibody neutralisation. However, the most effective protective defence against HIV-1 transmission may potentially be the combination of HIV-1-specific cytotoxic T-cell responses and anti-HIV-specific antibody responses. So far, a series of broadly NAbs to HIV-1 has been isolated from HIV infected individuals, but attempts to elicit this type of antibodies in animal models using HIV-envelope based immunogens failed to raise the desired broadly reactive NAbs. In addition, the compartmentalisation of the immune response in the systemic and mucosal surfaces has been repeatedly documented with a skewed distribution: antibodies can be present in the blood primarily as immunoglobulin G (IgG) isotype or in the mucosal compartment as IgA isotype. In most cases, the first port of entry for HIV is through the mucosa during sexual contact (above 80 % of HIV infections therefore having mucosal IgA as a first line of defense is especially important. The objective of this proposal is to develop a variety of different next-generation HIV-1 envelope immunogens that are aimed at eliciting high-titre and broadly NAb responses. Clearly the envelope antigens of the virion and of infected cells represent the most susceptible targets to inactivation of HIV infectivity by antibodies. Considering the fact that, to date, despite the huge amount of effort, no preventive HIV-1 vaccine is available that elicits broadly NAbs, it is obvious that this is a very challenging task. However, we believe that the pipeline that we will establish, in which new immunogens and innovative approaches on immunogen delivery and adjuvant development and application will be continuously assessed in animals, is vital to our success in halting the epidemic. Some HIV-1 infected individuals have HIV-1 specific broadly neutralising activity in their serum, providing a fundamental proof of principle that the desired NAbs can indeed be elicited in vivo. We believe that viral variants with specific envelope properties / sequences must have elicited these responses and may elicit these responses again in humans if formulated into an appropriate vaccine immunogen. We will focus on samples both from adults and from children. Transmission of HIV from mother to child occurs in the presence of maternal antibodies, albeit mostly non-neutralising, in the presence of a still developing immune system. Thus, HIV-1 envelope proteins isolated from paediatric samples may have unique immunogenic characteristics as they are representative of recently transmitted viruses yet evolving in the absence of strong humoral immune pressure in the child, which may allow these viruses to expose neutralisation epitopes. Thus characteristics of these paediatric viruses may be highly relevant for vaccine design. HIV-1 envelope genes from patients with HIV specific broadly neutralising activity will thus be cloned, sequenced and screened for functionality and sensitivity to neutralisation. A genetic and functional database will be created which will provide unprecedented insights into specific markers for neutralisation and the elicitation of NAb responses. Modifications that aim to enhance immunogenicity for inducing broadly neutralising specificity will be applied to envelope fragments, and tested in rabbits, to determine antibody titres and neutralising responses. In brief, we propose to develop a variety of strategies for eliciting broadly NAbs directed against the viral envelope proteins (the env genes). Using primarily envs from patients who have successfully mounted a bNAb response we will develop methods that boost the immunogenicity of gp120 and gp41, and aim at directing the focus of the immune response to critical conserved sites. We will also: 1. engineer CD4 binding site mutants that will prevent binding of gp120 to CD4 in vivo, that do not interfere with antibody recognition of this region; 2. create stable trimers that present gp120 in a context most similar to the native viral spike; 3. express new chimeric glycoproteins presenting broadly neutralising CD4i (CD4-induced) epitopes; 4. optimise gp41 subunit immunogens capable of inducing HIV neutralising IgA; and 5. hone the antibody targeting to conserved sites by immunisation with mixtures of several different gp140 proteins. We will test this newly developed immunogens expressed on VLPs and / or virosomes with promising new adjuvant formulations in a prime-boost regimen in rabbits. For HIV-1, particle-based vaccine strategies have promise to elicit high-titre, long-lived, humoral and cellular immune responses to a diverse number of viral epitopes. The use of particle-based immunogens is a proven effective vaccination strategy, as shown for the Human Papillomavirus. Although virosomes and VLPs can induce strong immune responses in the absence of adjuvants, it may be even further optimised by adjuvants that are specific for the deoxyribonucleic acid (DNA) prime, the protein boost, or for directing peripheral or mucosal immunity. In this study, a panel of compounds will undergo comprehensive analysis in different prime-boost immunisation schedules with different novel adjuvants. Promising combinations of immunogens and adjuvants will be tested in a non-human primate (NHP) model to assess the immune response and then challenged with a suitable HIV / Simian immunodeficiency virus (SIV) live virus. In summary, the following key scientific topics are our goals: - cloning and sequencing HIV-env genes, and development of a genetic and functional (sensitivity to antibodies) env database; - high-throughput cloning, expression and purification of HIV env genes that in potential may elicit broadly Nabs and the creation of env protein arrays; - antigen optimisation and identification of novel neutralisation-sensitive epitopes as HIV-1 vaccine candidates; - rational mutagenesis of env aimed at de-protecting highly conserved neutralisation epitopes that are kept in a cryptic conformation or at stabilising env in a conformation that optimally presents the CD4-binding site; - novel strategies for inducing inter-clade and intra-clade cross-reactive responses; - optimisation of antigen targeting to the correct cells of the immune system; - novel adjuvants for Th2 response and delivery strategies for mucosal routes; - optimisation of immunisation routes and prime-boost schedules; - exploitation of the knowledge on the mechanisms of HIV mucosal infection and mucosal immunity for the development of safe and effective vaccines capable of preventing HIV-1 infection; - development of an immunological platform with standardised procedures. Project results: Screening of the neutralising activity was performed in more than 500 subjects in early and chronic phase of the disease: more than 400 HIV-1-infected adults, 30 HIV-1-infected mothers and 23 HIV-1-infected children, 20 HIV-2 infected and 11 HIV-1/2 dual-infected adults. bNAbs activity was identified in approximately 25 % of HIV-1 infected subjects by 2 to 4 years from infection. HIV-2 infected individual had higher and broader intratype NAbs than HIV-1 infected individuals. The IgA fraction of the plasma also contributed to the neutralisation in HIV-2 infection. Studies of the B cell populations of HIV-1 infected subjects showed clearly that aberrant and exhausted B cell subpopulations were appearing early after infection and did correlate with the viremia of the individuals. Memory B cells, when decreased during infection, did not recover despite control of viremia with effective antiretroviral treatment. Furthermore we showed that soluble CD27, a marker of immune activation, may act to enhance IgG production and differentiation of activated memory or recently antigen-experienced B cells, thus providing an activation signal to antigen-experienced B cells. HIV envelopes genes from primary viruses obtained early after infection in each patient, who had successfully mounted bNAbs, were cloned and as today more than 1800 clones sequenced. A novel method was used for understanding the relationship between env sequence and the presence of bNAbs. Machine learning was used with iterative parameter selection using a heuristic algorithm to construct high-order models of bNAbs-prediction. This detailed and systematic approach gave rise to an informatic database containing genetic and functional information of the envelopes to determine the viral characteristics associated with induction of bNAbs. Accordingly, 7 viral envelopes were selected and expressed as trimeric soluble proteins for immunogenicity and antigenicity screening in mice and rabbits. Sequences from four patients and one envelope mutant were also selected to be forwarded for processing into delivery systems and expressed as trimeric envelope-complexes on the surface of HIV-gag VLPs (gag-VLPs), to further improve immunogenicity. In parallel new strategies with stably transfected insect cell line were designed to improve and optimise VLP production and expression of glycoproteins on the surface. The research on the delivery systems had a parallel track using ready-to-go products available to NGIN for testing and also for improving the technology of two additional delivery systems: heterologous-VLP and virosomes. Expression of gp41 peptides gave promising results. Whereas expression of gp120 / gp140 proteins needed further evaluation for the development of alternative grafting procedures. Novel adjuvant formulations were evaluated such as CCL28, the pFlic, DDA / TDB (CAF01) and cholera toxin subunit B (CT-B). Two of those, pFlic and DDA / TDB, were testing in rabbits in association with DNA and proteins, respectively. DDA/TDB showed a clear enhancement of the neutralising antibody response when administered with gp140 trimers in rabbits, whereas pFlic and CT-B did not show any considerable enhancement. CCL28 demonstrated to be a good inducer of mucosal antibody responses in mice, and thus shows to be promising for further animal studies. In total 40 experiments in rabbits have been undertaken and immunogenicity assessed. DNA or trimeric proteins embedded or not into gag-VLPs, heterologous-VLP or virosomes were used either alone or in prime-boost approach. The product pipeline of NGIN provides evidence of solid heterologous NAb responses, mainly restricted to tier 1 viruses, induced in immunised rabbits. In specific, NAbs: - can be detected two weeks after the first boost using DNA or trimer protein immunisation protocols in the rabbit serum; - require elicitation of protein-binding antibody titres in the range or above approximately 105; - are elevated after boosting, while later decline, but not to the same extent as protein-binding antibody titres; - had highest titres when immunogens included trimer gp140 proteins at a minimum protein dose of 100 µg and adjuvant; - can be elevated after DNA priming followed by trimer protein boost. The most promising reagents were forwarded for administration to cynomologous macaques with a prime-boost regimen. NGIN pursued three immunisation procedures with the prime-boost being: 1. intra-subtype (B DNA - B protein); 2. inter-subtype (B DNA - A protein); and 3. inter-virus group (HIV DNA - SIV protein). The third approach was introduced as to confirm the hypothesis that boosting heterologous viruses would favour antibody responses to highly conserved regions. Testing of NAb elicited in immunised macaques revealed that NAb responses were restricted to tier 1 viruses. NAbs were already detected in serum after DNA immunisation, and were enhanced to higher titers after gp140 trimer protein boost. No mucosal NAbs could be detected. The immunological platform of NGIN established standardised and well-controlled neutralisation assays with humoral and mucosal materials. It has continued the collaboration with the EUROPRISE NeutNet platform to exchange experience on neutralisation assays and related issues. HPA has provided a wide range of research reagents and reference materials as documented by the shipments of thousands of different reagents. Robust and continuous management of the project along with balanced attention to both scientific and administrative aspects have proven to be successful to proceed in a positive and timely manner. The reserved area of the NGIN website (see http://www.ngin.eu online) has proven pivotal for sharing information, protocols and results amongst participants. The website has also a public domain, which informs on the project features and provides information on NGIN related publications and issues of general interest. Potential impact: NGIN has represented a tight collaboration of 13 institutions as well as 3 small and medium-sized enterprises (SMEs) throughout Europe. It has involved about 60 scientists and in specific has given the possibility to train a large number of young researchers and Doctor of Philosophy (PhD) students. Eleven PhD students completed their training during NGIN. The close contact and synergy with other ongoing projects funded by European Commission (EC), for example EUROPRISE, AUTOCELL, and NEUTNET, has fostered the training of researcher and exchange of scientific knowhow. NGIN had a product pipeline which has given rise to a set of new envelope-based immunogens, shown to be immunogenic in rabbits and in non-human primates. Thus, NGIN is providing new products apt to be forwarded for challenge studies in non-human primates. We have provided new or have refined delivery systems. NGIN has explored known adjuvants such as DDA / TDB, which are already used for human trials of tuberculosis (TB) and HIV infection prevention, and also new adjuvants, such as CCL28, which should be forwarded for further studies to test its potential for induction of mucosal immunity. Dissemination efforts within the consortium have been impressive with 52 peer-reviewed publications and at least 78 dissemination activities (participation to topic-relevant conferences, workshops, discussion of theses). An important feature of this project has been the balanced gender distribution of the scientists involved, both at the apical and basal positions: moreover eleven female PhD students finalised their work leading to successful thesis discussion. Protection from microbes through vaccination of the host remains the most effective way of protection from infection for the individual and society. The present project has increased the current knowledge in immunogen design and may lead to improved HIV vaccination protocols and novel adjuvant strategies. The outcome of this research project has ultimately contributed to the benefit of the health of European citizens. List of websites: http://www.ngin.eu Related documents Final Report - NGIN (Next generation HIV-1 immunogens inducing broadly reactive neutralising antibodies)
