An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (nAbs) that possess a unique ability to bind to authentic Env trimers. It is logical, therefore, that these trimers may be able to elicit nAbs in a vaccine setting. In addition to trimers, however, particles bear non-functional Env that appears to dominate Ab responses and dampen or delay nAb development. We will test the hypothesis that anti-trimer responses to particulate vaccines are improved when unfettered by non-functional Env.
Our Specific Aims are:
Specific Aim 1 : To Investigate the effect of V1V2 and V3 loop mutations on lateral trimer stability. One strategy to eliminate undesirable Ab targets on VLPs may be to laterally stabilize trimers (i.e. between adjacent gp120/gp41 protomers) by an inter-molecular disulfide bridge. To assist in the placement of cysteines, we will perform targeted alanine scanning mutagenesis of authentic Env trimers. We have preliminary evidence that screening by BN-PAGE makes the identification of laterally unstable mutants feasible. Available data suggests that variable loop interactions may stabilize neighboring gp120 subunits.
Specific Aim 2 : To laterally stabilize authentic trimers by introducing an inter-gp120 disulfide bond. We will make pairs of cysteine substitutions in V1V2 and V3 loops to try to introduce a disulfide bridge, termed """"""""SOSVV"""""""", focusing on positions identified in Aim 1. To test whether a V-V disulfide bridge is present, we will evaluate trimer stability to ionic detergents and reducing agents. Native PAGE binding studies will be used to assess trimer authenticity as indicated by nAb binding exclusivity. We will also examine the ability of the SOSVV trimers to function in infection and the stability of SOSVV mutants expressed as a soluble gp140.
Specific Aim 3 : To evaluate the ability of unfettered authentic trimers to elicit nAbs in rabbits. If SOSVV forms stable trimers with no non-functional Env contamination, we will test their immunogenicity in rabbits. If neutralizing responses fail to develop, we will try higher doses and hyperimmunization. Another possibility would be to use VLPs complexed with IgG to augment nAb responses. Contingent immunogens will either be soluble SOSVV or VLP immunogens in which non-functional targets are masked by species-matched IgG. Results will drive successive immunizations to solve problems and amplify neutralizing responses.
Specific Aim 4 : To augment antibody responses to authentic trimers in macaques. The R33 phase has 3 main components. First, we will adapt our immunogens for macaques. Macaques offer both opportunities and challenges. For example, broad nAbs can be generated in SHIV-infections. However, Env-based immunogens may engage endogenous primate CD4, leading to the elicitation of non-neutralizing Ab specificities. Therefore, we will evaluate CD4 binding knockout trimers. We will immunize two groups of 12 macaques and challenge the second group with a heterologous SHIV. Second, we will try to improve nAb titer and breadth in rabbits by various strategies. Third, we will try to improve the production, purification and quality of VLPs.

Public Health Relevance

We will investigate HIV virus-like particles as immunogens for eliciting neutralizing antibodies in rabbits and macaques. Antibody responses to non-functional forms of Env on virus-like particles may dampen or delay the emergence of neutralizing responses. We will investigate the possibility to link adjacent gp120 protomers within authentic trimers as a way to eliminate non-functional Env and amplify neutralizing responses generated against authentic trimers.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants Phase II (R33)
Project #
Application #
Study Section
Special Emphasis Panel (NSS)
Program Officer
Li, Yen
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Torrey Pines Institute for Molecular Studies
Port Saint Lucie
United States
Zip Code
Cale, Evan M; Gorman, Jason; Radakovich, Nathan A et al. (2017) Virus-like Particles Identify an HIV V1V2 Apex-Binding Neutralizing Antibody that Lacks a Protruding Loop. Immunity 46:777-791.e10
Luo, Yang; Jacobs, Erica Y; Greco, Todd M et al. (2016) HIV-host interactome revealed directly from infected cells. Nat Microbiol 1:16068
Kwon, Young Do; Pancera, Marie; Acharya, Priyamvada et al. (2015) Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env. Nat Struct Mol Biol 22:522-31
Crooks, Ema T; Tong, Tommy; Chakrabarti, Bimal et al. (2015) Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site. PLoS Pathog 11:e1004932
Huang, Jinghe; Kang, Byong H; Pancera, Marie et al. (2014) Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface. Nature 515:138-42
Tong, Tommy; Crooks, Ema T; Osawa, Keiko et al. (2014) Multi-Parameter Exploration of HIV-1 Virus-Like Particles as Neutralizing Antibody Immunogens in Guinea Pigs, Rabbits and Macaques. Virology 456-457:55-69
Tong, Tommy; Osawa, Keiko; Robinson, James E et al. (2013) Topological analysis of HIV-1 glycoproteins expressed in situ on virus surfaces reveals tighter packing but greater conformational flexibility than for soluble gp120. J Virol 87:9233-49
Löving, Robin; Sjöberg, Mathilda; Wu, Shang-Rung et al. (2013) Inhibition of the HIV-1 spike by single-PG9/16-antibody binding suggests a coordinated-activation model for its three protomeric units. J Virol 87:7000-7
Selhorst, Philippe; Grupping, Katrijn; Tong, Tommy et al. (2013) M48U1 CD4 mimetic has a sustained inhibitory effect on cell-associated HIV-1 by attenuating virion infectivity through gp120 shedding. Retrovirology 10:12
Bontjer, Ilja; Melchers, Mark; Tong, Tommy et al. (2013) Comparative Immunogenicity of Evolved V1V2-Deleted HIV-1 Envelope Glycoprotein Trimers. PLoS One 8:e67484

Showing the most recent 10 out of 14 publications