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Science Spotlight

Multi-step HIV-1 vaccines

From the Vaccine and Infectious Disease Division and the Chan Zuckerberg Biohub.

A vaccine for human immunodeficiency virus, type 1 (HIV-1, is urgently needed to reduce the global incidence of transmission. Therefore, HIV-1 vaccine development is an enormous focus within global health research. One vaccine strategy involves eliciting antibodies capable of neutralizing of broad range of HIV-1 viruses, termed broadly neutralizing antibodies (bnAbs). VRC01 antihodies are a class of bnAbs which bind the CD4 binding site of the HIV-1 envelope (Env). They have been isolated from infected patients, but not yet elicited by vaccination. Recombinant HIV-1 Env proteins have been designed which bind and activate the unmutated (germline) forms of VRC01 (glVRC01) B cell receptors (BCR) in mice, but the resultant antibodies cannot bind their epitope on the virus itself because of steric hindrance from glycans at the conserved position N276. This creates a challenge wherein the germline-targeting immunogen that can bind glVRC01 lacks N276 glycan, and the elicited by immunization antibodies then in turn, cannot neutralize the HIV-1 virus due to steric inhibition imposed by the N276 glycan.

A strategy to overcome this problem uses sequential immunizations to guide antibody maturation in a targeted, step-wise manner. Rachael Parks, a PhD candidate in the Stamatatos lab (Vaccine and Infectious Disease Division), recently published work in Cell Reports that uses this framework to initially activate glVRC01 expressing B cells with a germline-targeting immunogen that lacks N276 and secondarily boost the B cells responses with an immunogen containing N276 glycan. The Stamatatos lab previously designed a successful "germline-targeting" immunogen for activating naïve B cells expressing glVRC01 BCRs in vitro, termed 426c Core. 426c Core is a recombinant protein derived from the clade C 426c Env. To first confirm that the 426c Core could elicit VRC01 Abs in vivo, Parks immunized transgenic mice—a strain with the human allele that encodes the heavy chain component of the VRC01-class antibody knocked in—the authors isolated Env-specific B cells from mice two weeks post-immunization and sequenced their BCR. Interestingly, they found that the B cells stimulated by 426c Core expressed BCRs with mutations present in human VRC01-class antibodies. The authors then resolved the crystal structure of 426c Core when bound to a corresponding antibody and found that this interaction structurally mirrored human VRC01-class antibody binding. Together, these experiments confirmed that 426c Core could elicit VRC01-like antibodies in vivo.

Parks and colleagues next tested the elicited antibodies for neutralization in vitro against HIV-1. They evaluated antibody neutralization against wildtype (WT) virus either containing or lacking the glycan at N276. As expected, the antibodies were only able neutralize virus that lacked the N276 glycan. However, when the authors tested the antibodies for binding against autologous and heterologous Env Core proteins that have intact N276 glycan but lack other sites of steric hindrance, they observed binding. This led the authors to hypothesize that a second, boosting immunization with a heterologous Core Env expressing glycan at position N276 would expand the intermediate population of B cells into those capable of binding Env and neutralizing HIV-1 virus with the N276 glycan intact. To this end, four weeks after immunizing mice with 426c Core, the authors administered a boost immunization with WT Core Env, which is glycosylated at position 276. They then isolated and sequenced Env-specific B cells and generated 15 antibodies from the resulting paired heavy and light chain BCR sequences. Compared to antibodies elicited from the prime alone, antibodies elicited from the boost contained more mutations in the BCR and successfully bound trimeric WT Env. Since antibodies generated from the prime immunization were only able to bind to trimeric Env lacking the N276 glycan, this suggested that the second immunization further drove antibody maturation so that the post-second immunization antibodies could bypass the N276 glycans. This was confirmed by Ab-Env immunoprecipitation and Mass Spec analysis of the immunoprecipitated Env. Indeed, one of the antibodies elicited following the second immunization neutralized WT 426c virus, something that human glVRC01 antibody cannot do.  These results led the authors to conclude that VRC01-like antibodies resulting from prime-boost immunizations with the above two immunogens could accommodate the steric hindrance imposed by N276 glycan and that this immunization scheme is effective in guiding the evolution of VRC01-like neutralizing antibodies. 

Graphical abstract of two-step immunization scheme. Humanized mice are immunized with 426c core immunogen, resulting in VRC01-like antibodies which can bind HIV Env lacking glycan at N276. A boosting immunization with a second immunogen containing glycans at N276 further direct antibody development to allow for neutralization of complete HIV Env.
Graphical abstract of two-step immunization scheme. Humanized mice are immunized with 426c core immunogen, resulting in VRC01-like antibodies which can bind HIV Env lacking glycan at N276. A boosting immunization with a second immunogen containing glycans at N276 further direct antibody development to allow for neutralization of complete HIV Env. Image provided by Rachael Parks.

This achievement represents a significant advancement in the field of bnAbs against HIV-1, as these experiments reveal it is possible to guide glVRC01 into a more mature antibody that can overcome steric hindrance to successfully neutralize WT HIV-1 virus. Parks summarized her work: “This study identifies a prime-boost immunization scheme that activates the unmutated BCRs that give rise to a class of HIV-1 bnAbs called VRC01-class, and guides their evolution towards an early neutralizing stage,” Parks explained.  Going forward, “follow up work aims at further enhancing the neutralizing potential of these antibodies. The immunogens identified in this study will be evaluated in humans (in a phase I clinical trial at the HIV Vaccine Trials Network) where the VRC01-class BCRs are expressed at a lower frequency.”

Parks KP, MacCamy AJ, Trichka J, Gray M, Weidle C, Borst AJ, Khechaduri A, Takushi B, Agrawal P, Guenaga J, Wyatt RT, Coler R, Seaman M, LaBranche C, Montefiori DC, Veesler D, Pancera M, McGuire A, Stamatatos L. 2019. Overcoming Steric Restrictions of VRC01 HIV-1 Neutralizing Antibodies through Immunization. Cell Reports. 2019 Dec 3;29(10):3060-3072.e7. doi: 10.1016/j.celrep.2019.10.071.


This work was supported by the National Institutes of Health, the Pew Charitable Trust, the Burroughs Wellcome Fund, the Arnold and Mabel Veckman Cryo-EM Center, and the University of Washington Proteomics Resource.

UW/Fred Hutch Cancer Consortium member Andrew McGuire contributed to this work.