An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (nAbs) that possess aunique ability to bind to authentic Env trimers. It is logical, therefore, that these trimers may be able to elicitnAbs in a vaccine setting. In addition to trimers, however, particles bear non-functional Env that appears todominate Ab responses and dampen or delay nAb development. We will test the hypothesis that anti-trimerresponses 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. Onestrategy to eliminate undesirable Ab targets on VLPs may be to laterally stabilize trimers (i.e. between adjacentgp120/gp41 protomers) by an inter-molecular disulfide bridge. To assist in the placement of cysteines, we willperform targeted alanine scanning mutagenesis of authentic Env trimers. We have preliminary evidence thatscreening by BN-PAGE makes the identification of laterally unstable mutants feasible. Available data suggeststhat variable loop interactions may stabilize neighboring gp120 subunits.
Specific Aim 2 : To laterally stabilize authentic trimers by introducing an inter-gp120 disulfide bond. Wewill 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 willevaluate trimer stability to ionic detergents and reducing agents. Native PAGE binding studies will be used toassess trimer authenticity as indicated by nAb binding exclusivity. We will also examine the ability of theSOSVV 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 SOSVVforms stable trimers with no non-functional Env contamination, we will test their immunogenicity in rabbits. Ifneutralizing responses fail to develop, we will try higher doses and hyperimmunization. Another possibilitywould be to use VLPs complexed with IgG to augment nAb responses. Contingent immunogens will either besoluble 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 3main components. First, we will adapt our immunogens for macaques. Macaques offer both opportunities andchallenges. For example, broad nAbs can be generated in SHIV-infections. However, Env-based immunogensmay 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 challengethe second group with a heterologous SHIV. Second, we will try to improve nAb titer and breadth in rabbits byvarious strategies. Third, we will try to improve the production, purification and quality of VLPs.
We will investigate HIV virus-like particles as immunogens for eliciting neutralizing antibodies in rabbits andmacaques. Antibody responses to non-functional forms of Env on virus-like particles may dampen or delay theemergence of neutralizing responses. We will investigate the possibility to link adjacent gp120 protomers withinauthentic trimers as a way to eliminate non-functional Env and amplify neutralizing responses generatedagainst authentic trimers.
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