Document text
Principal Investigator: paolo lusso
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2024
Award: $1,410,429
Funding agency: National Institute of Allergy and Infectious Diseases
The development of an HIV vaccine has been hampered by unprecedented challenges, primarily due to the unique properties of the HIV-1 envelope (Env) trimer, which features an extraordinary assortment of immune-evasion tactics, including antigenic variation, heavy glycosylation of exposed surfaces and conformational masking of key neutralization sites. Further insights into the complex structure-function relationships in the HIV-1 Env trimer and its protective shield may be critical to guide the rational design of a protective vaccine and other immunoprophylaxis measures.
1) Enhancement of broadly HIV-neutralizing antibodies.
Passive transfer of bNAbs is a promising alternative to ART for HIV-1 prevention and treatment. We previously reported the discovery of a second CD4-binding site in the HIV-1 Env trimer, that we defined as CD4-binding site 2 (CD4-BS2), which opened new perspectives for HIV vaccine and therapy (Liu et al., NSMB 2018). We also identified selected anti-CD4-BS antibodies, such as VRC03 and VRC06, which mimic the quaternary-binding mode of CD4 and establish contact with two adjacent gp120 protomers via an extended loop in their heavy chain framework region 3 (FR3). Thus, we rationally engrafted the extended FR3 loop of VRC03 onto different CD4-supersite bNAbs and developed chimeric antibodies with enhance activity against a wide panel of global HIV-1 strains (Liu et al. 2019). We applied this strategy to a new family of anti-CD4bs bNAbs (i.e., the N49 lineage) and the resultant chimeric antibodies bound the Env trimer with a higher affinity than their parental forms and displayed increased neutralizing activity against a global panel of HIV-1 Envs, with a remarkable mean half-maximal inhibitory concentration below 0.01 g/mL against a wide panel of 86 global HIV-1 strains. Due to their increased potency associated with reduced autoreactivity and prolonged in vivo half-life, FR3-loop-chimeric bNAbs are under consideration for clinical use in HIV-1 prevention and treatment. In parallel studies, we are also trying to design more potent chimeric antibodies by screening recombinant libraries of FR3 segments to be inserted into the bNAb VRC07.
2) Design of Soluble HIV-1 Envelope Trimers Free of Covalent gp120-gp41 Bonds with Prevalent Native-Like Conformation. Soluble HIV-1 envelope trimers may serve as effective vaccine immunogens. The widely utilized SOSIP trimers have been paramount for structural studies, but the disulfide bond they feature between gp120 and gp41 constrains intersubunit mobility and may alter antigenicity. Here, we report an alternative strategy to generate stabilized soluble Env trimers free of covalent gp120-gp41 bonds. Stabilization was achieved by introducing an intrasubunit disulfide bond between the inner and outer domains of gp120, defined as interdomain lock (IDL). Correctly-folded IDL trimers displaying a native-like antigenic profile were produced for HIV-1 envelopes of different clades. Importantly, the IDL design abrogated CD4 binding while not affecting recognition by potent neutralizing antibodies to the CD4-binding site. By cryo-electron microscopy, IDL trimers were shown to adopt a closed prefusion configuration, while single-molecule FRET analysis documented a high prevalence of native-like conformation. Thus, IDL trimers may be promising candidates as vaccine immunogens.
3.Development and pre-clinical evaluation of a VLP-forming mRNA vaccine against HIV-1.
a. Gag processing by retroviral protease enhances the immunogenicity of VLP-forming mRNA vaccines. Although co-expression of Env with Gag leads to the assembly and release of VLPs, in the absence of the viral protease Gag remains uncleaved and only immature VLPs are formed. Since mature VLPs are believed to be better immunogens than immature VLPs, we aimed at introducing the viral protease (pro) into our env-gag mRNA vaccine platform. Since the use of pro mRNA induced toxicity and scarce VLP generation, we decided to employ the physiological mechanism utilized by HIV-1, which transcribes the pro gene as part of the long gag-pol mRNA transcript, resulting in the production of high titers of well-shaped mature VLPs using env:gag:gag-pol at a ratio of 1.5:1:0.1.
b. Evaluation of a protease-supplemented HIV-1 mRNA vaccine in VRC01gl knock-in mice. To evaluate the efficiency of our protease-enriched triuple mRNA formulation (env+gag+gag-pol), we immunized knock-in trangenic mice bearing the germline (gl) version of the bNAb VRC01. The mice developed extremely high levels of specific B-cell activation as well as trimer-binding and neutralizing antibodies in serum. MAbs have been cloned from these mice and are being characterized for function and affinity maturation.
c. Design and pre-clinical evaluation of a VLP-forming HIV-1 mRNA vaccine in macaques. We designed an mRNA vaccine with the following features: i) use of mRNA as a vehicle to endogenously express full-length membrane-bound Env glycoproteins decorated with native N-linked glycosylation; ii) co-expression of Env with Gag and Gag-Pol to induce the in vivo production of mature virus-like particles (VLP), which closely mimic native viral particles produced by HIV-1 infection; iii) initial priming with an Env capable of engaging germline bNAb precursors; iv) intensive heterologous boosting with tier-2 Envs from different clades (A,B,C) to selectively expand antibody responses against shared bNAb epitopes. Three pre-clinical immunization-challenge trials have been conducted in Rhesus macaques. In all 3 studies, the vaccine was highly immunogenic and induced not only high levels of trimer-binding antibodies, but also broad-spectrum tier-2 neutralizing antibodies, albeit at relatively low titers. Importantly, vaccinated animals in all 3 studies were significantly protected from repeated low-dose mucosal challenges with a difficult-to-neutralize heterologous tier-2/3 SHIV (SHIV-AD8) with calculated per-exposure risk reductions of ~80%. These results provided evidence that a multiclade VLP-forming mRNA vaccine can be effective in protecting against heterologous tier-2 infection.
Terms: <AIDS Virus><AIDS/HIV><Ab-mediated immunity><Ab-mediated protection><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Adopted><Affinity><Animals><Antibodies><Antibody Response><Antibody immunity><Antibody protection><Antibody-mediated protection><Antigen Variation><Antigenic Determinants><Antigenic Variability><Antigenic Variation><Antigens><B-Cell Activation><Binding><Binding Determinants><Binding Sites><Biological><Blood Serum><Clinical><Combining Site><Complex><Cryo-electron Microscopy><Cryoelectron Microscopy><Development><Dose><Electron Cryomicroscopy><Engraftment><Epitopes><Esteroproteases><Evaluation><Family><Formulation><Framework Regions><Gene Transcription><Generations><Genes><Genetic Transcription><Germ Lines><Glycoproteins><Goals><HIV><HIV Envelope Glycoprotein gp120><HIV Envelope Protein gp120><HIV Infections><HIV env Protein gp120><HIV therapy><HIV vaccine><HIV-1><HIV-1 vaccine><HIV-I><HIV/AIDS><HIV/AIDS Vaccines><HIV1><HIV1 vaccine><HTLV-III Infections><HTLV-III gp120><HTLV-III-LAV Infections><Half-Life><High Prevalence><Human Immunodeficiency Virus Type 1><Human Immunodeficiency Viruses><Human T-Lymphotropic Virus Type III Infections><Human immunodeficiency virus 1><Immune Evasion><Immunization><Immunize><Infection><KI mice><Knock-in><Knock-in Mouse><Knowledge><LAV-HTLV-III><Length><Libraries><Link><Lymphadenopathy-Associated Virus><M mulatta><M. mulatta><Macaca><Macaca mulatta><Macaque><Measures><Membrane><Messenger RNA><Metabolic Glycosylation><Mice><Mice Mammals><Modification><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Mucosa><Mucosal Tissue><Mucous Membrane><Murine><Mus><Peptidases><Peptide Hydrolases><Physiologic><Physiological><Prevention><Production><Property><Protease Gene><Proteases><Proteinases><Proteolytic Enzymes><Protomer><RNA Expression><RNA vaccine><RNA-based vaccine><Reactive Site><Receptor Protein><Recombinants><Reporting><Rhesus Macaque><Rhesus Monkey><Risk><Rodent><Rodentia><Rodents Mammals><SHIV><Serum><Shapes><Site><Structure><Structure-Activity Relationship><Surface><Technology><Toxic effect><Toxicities><Transcript><Transcription><Vaccinated><Vaccine Design><Vaccines><Viral><Virus-HIV><Virus-like particle><activated B cells><antibody-mediated immunity><autoimmune reactivity><autoreactivity><biologic><chemical structure function><chimeric antibody><conformation><conformational><conformational state><conformationally><conformations><cryo-EM><cryoEM><cryogenic electron microscopy><design><designing><developmental><disulfide bond><glycosylation><gp120><gp120 ENV Glycoprotein><gp120(HIV)><human immunodeficiency virus vaccine><immune evasive><immunogen><immunogenic><immunogenicity><immunoprophylaxis><improved><in vivo><insight><knockin><knockin mice><mRNA><mRNA vaccine><mRNA-based vaccine><membrane structure><natural antibodies><neutralizing antibody><novel><particle><pre-clinical><pre-clinical evaluation><preclinical><preclinical evaluation><prevent><preventing><rational design><receptor><screening><screenings><simian HIV><simian human immunodeficiency virus><single-molecule FRET><single-molecule fluorescence resonance energy transfer><smFRET><structure function relationship><vaccine platform><virus-like nanoparticles><viruslike particle>