Empowering nucleic acid technology to generate highly conformational and functional vaccines against HIV in vivo

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Rapid and large-scale deployment of COVID-19 mRNA vaccines highlights the potential utility of developing nucleic acid vaccines (such as RNA and DNA vaccines) against infectious diseases, including HIV-1. However, as compared to SARS-CoV-2, HIV-1 vaccines pose some unique challenges for induction of neutralizing antibodies (NAbs), a potential correlate for protection.  For HIV, the induction of such antibodies requires presentation of trimeric and highly conformational epitopes to the immune system.   Whether encoded nucleic acid vaccines can enable direct in vivo production of structurally relevant antigens which retain a critical antigenic profile has not yet been demonstrated.  Additionally, it was previously reported that for HIV that robust Tier 2 NAbs cannot be induced in mice due to a lack of the required antibody repertoire1.  This limitation drove immunization studies to be performed in larger mammals such as rabbits/ NHPs, inadvertently slowing down and increasing the costs of preclinical HIV-1 vaccine studies, as well as limiting our immune tools for advancing novel HIV vaccine formulations. 

To that end, we set out to design a rigorous assay to evaluate antigenic profiles of in vivo produced vaccines2. For our proof-of-concept, we turned to an immunogen designated MD39, which is based off BG505.SOSIP.664, and assembles in vitro as Native Like Trimers (NLTs) that mimic viral envelopes on infected cells3,4. The Nucleic Acid sequence for this trimer was genetically  optimized and developed in a DNA delivery cassette.   This construct produces self assembling NLT in laboratory cells with high efficiency and stability.  We next delivered the DNA encoded sequences efficiently into the muscles of mice using the adaptive electroporation technology.  We sought to interrogate the nature of the delivered NLT inside the animals.   We are fortunate in the HIV space there exists a diverse library of broadly neutralizing antibodies (bNAbs) that bind to various well-characterized epitopes on the HIV Envelope. We were excited to find that in vivo produced antigens, just like proteins produced in the cell lines, preferentially bind to trimer-specific bNAbs but not to poorly-assembled, monomer-specific non-neutralizing antibodies (nNAbs).  This outcome was confirmed using both immunofluorescence and our in-house assay called ACTIVE, or Antigen Conformation Tracing In Vivo by ELISA.

As a result of the in vivo assembly, we observed that the vaccines produced directly in the host as NLTs were highly functional. DNA-encoded NLT (pMD39) not only was able to induce more robust T cell responses in mice than corresponding protein vaccination, but we also found that T follicular helper and germinal center B cell to be higher. More strikingly, we found that DNA vaccination uniquely induced Tier 2 autologous neutralizing antibody responses in mice, contrary to the previous paradigm that mice lack the required repertoire to mount such response, highlighting the functional importance of in vivo assembly of structured antigens. 

We were curious to explore how such how these murine antibodies could mediate neutralization, and decided to test these antibodies against various engineered pseudoviruses. These pseudoviruses harbor specific mutations in epitopes that were previously mapped in rabbits and macaques, such that if our antibodies lose neutralization against one specific variant, we know that these murine Abs are likely directed towards such epitope. In this manner, we determined that murine MAbs induced by our vaccination regimen predominantly targeted the C3/V5 epitope on the HIV Envelope, a neutralizing epitope that was previously only described in Rhesus macaques5.

We thought further structural validation was important to provide unequivocal description of the epitope specificities of these induced antibodies. We collaborated with Dr. Jesper Pallesen at Indiana University to obtain  CryoEM structure of the Env-Fab complex of up to 3.8Å in resolution. We observed extensive engagement of our antibodies with the C3/V5 site on HIV Env; in particular, the HCDR2 of our antibody was in close contact with R456 residue at the CD4 binding site.  These observed interactions not only affirm our prior findings in the engineered pseudovirus assays but also provides a direct mechanism for induction of neutralization. We are hoping to use this detailed structural information on this neutralizing epitope to inform design of next-generation HIV vaccines that can potentially generate more breadth.

The advent of COVID-19 mRNA vaccines highlights potential of the nucleic-acid based vaccine technology6. Through bypassing the complex and specialized production and purification pipelines (as in the case of protein-based vaccines), nucleic acid-based vaccines can be rapidly scaled and deployed for the current and possible future pandemics. However, we do believe that it remains paramount that we carefully examine the antigenic profiles of vaccines produced in vivo, especially in the context of a highly challenging target such as HIV-1. We have, here and previously, demonstrated that DNA cassettes can be used to launch trimeric and nanoparticles de novo in the host2,7. The assays described here can also be generalized to examine other types of vaccines. Finally, we also believe that the observation that mice can be used in HIV-1 vaccine efforts is important; as rapid iterative evaluations of various structurally designed HIV-1 vaccine candidates are now possible.

References

1          Hu, J. K. et al. Murine Antibody Responses to Cleaved Soluble HIV-1 Envelope Trimers Are Highly Restricted in Specificity. J Virol 89, 10383-10398, doi:10.1128/JVI.01653-15 (2015).

2          Xu, Z. et al. Induction of tier-2 neutralizing antibodies in mice with a DNA-encoded HIV envelope native like trimer. Nature Communications 13, 695, doi:10.1038/s41467-022-28363-z (2022).

3          Steichen, J. M. et al. HIV Vaccine Design to Target Germline Precursors of Glycan-Dependent Broadly Neutralizing Antibodies. Immunity 45, 483-496, doi:10.1016/j.immuni.2016.08.016 (2016).

4          Xu, Z. & Kulp, D. W. Protein engineering and particulate display of B-cell epitopes to facilitate development of novel vaccines. Curr Opin Immunol 59, 49-56, doi:10.1016/j.coi.2019.03.003 (2019).

5          Zhao, F. et al. Mapping Neutralizing Antibody Epitope Specificities to an HIV Env Trimer in Immunized and in Infected Rhesus Macaques. Cell Rep 32, 108122, doi:10.1016/j.celrep.2020.108122 (2020).

6          Polack, F. P. et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med 383, 2603-2615, doi:10.1056/NEJMoa2034577 (2020).

7          Xu, Z. et al. In Vivo Assembly of Nanoparticles Achieved through Synergy of Structure-Based Protein Engineering and Synthetic DNA Generates Enhanced Adaptive Immunity. Adv Sci (Weinh) 7, 1902802, doi:10.1002/advs.201902802 (2020).

 

Ziyang Xu

Post Doctoral Fellow, University of Pennsylvania