**10. Promising vaccine strategies: eOD-GT8 60mer and gene therapy**

Additional BnAb-based vaccines currently being assessed are eOD-GT8 60 mer (protein nanoparticle derived from the VRC01 CD4-binding site) and gene therapy through a recombinant adeno-associated virus-mediated (rAAV) construct containing HIV BnAb genes [44].

This eOD-GT8 60mer delivered with AS01B adjuvant was tested in 2018 in an early clinical trial. This nanoparticle was originally studied in genetically modified knock-in mice and animal studies and designed to activate B-cell precursors to form BnAb. The results showed that 97% subjects produced precursor VRC01 IgG B cells, according to the preliminary data in early 2021 at the 2021 R4P conference. This is an important finding, because it provides some evidence that stimulation of rare B-cell precursors is possible. Further study is needed to validate this approach and determine that it works consistently [44].

A gene therapy approach to delivery BnAb genes utilizes the rAAV vector and selects genes like VRC01, b12, 4E10, 2F5, 2G12, 3BNC117, 10–1074, and 10E8. This strategy was assessed in rhesus macaques and led to undetectable viremia for three years. While this was incredibly positive, the animals developed antidrug antibody (ADA) responses. Further testing with nonhuman primates (NHP) yielded similar results, with a decline of a BnAb corresponding to ADA response. In the NHP study, these animals did have mucosal protection against the simian version of HIV but for a shorter duration (less than a month). This may have been due to only receiving one BnAb gene in the rAAV vector. The ADA response remains the main hurdle in this approach, provided that the BnAb gene combinations are validated [44].

These initial data from both the eOD-GT8 60 mer and rAAV vector with BnAb gene immunization strategies are encouraging. More research and trials are necessary to fully understand their potential to generate a prophylactic HIV-1 immunization.

## **11. Promising vaccine strategies: HIVcons immunogen-based vaccine**

A promising concept that may result in a prophylactic HIV-1 vaccine is one with a HIVcons immunogen basis. This design is a 778 amino acid insert created analyzing all clades through bioinformatics. The insert specifically focused on sequence conservation and did not use epitopes as a "string of beads" [13]. String of beads epitope design refers to a technique where short sequences of amino acids or spacers are added between epitopes in an attempt to allow the epitopes to be cleaved correctly [52]. Researchers creating HIVcons immunogen elected to not use the string of beads construct, because it could create irrelevant epitopes not present on HIV-1-infected cells. Additionally, this construct could skew the design toward frequent Caucasian HLA alleles [13].

While the HIVcons insert was successfully tested in multiple early clinical trials in two different vectors in both healthy volunteers and patients with HIV-1 infections, researchers had to redesign to create a "second-generation conserved mosaic tHIVconsvX" vaccine, utilizing the CdAdOx1 vector from simian adenovirus Y25. This was due to licensure issues with the previous vectors, but believed to be advantageous overall since the second-generation design was improved, as discussed by Tomas Hanke [13].

### **12. Promising vaccine strategies: mosaic immunogens**

Mosaic immunogens is another vaccine design that is showing promising results in clinical trials. These immunogens are bioinformatically optimized bivalent antigens derived from different clades of HIV-1 group M strain [53]. These sequences were then placed into MVA or Ad26 vectors, followed by a clade C gp140 protein boost. This was evaluated in the APPROACH study in East Africa, South Africa, Thailand, and the USA. The results indicated that the Ad26/Ad26+ high dose gp140 vaccinations produced Env-specific antibody responses and T cell responses in the vast majority of recipients (100%, 83% for each of these responses) [54].

Building off the APPROACH study data as well as TRAVERSE and ASCENT studies, two studies, Imbokodo (HVTN 705) and Mosaico (HVTN 706), were designed. Imbokodo was phase 2b efficacy clinical trial for women in Southern Africa evaluating a heterologous prime/boost of Ad26.Mos4.HIV and adjuvanted aluminum phosphate Clade C gp140 [6, 44]. In total, it has four mosaic antigens within the vector aimed at inducing an immune response. Preliminary results of Imbokodo showed no significant efficacy (25.2% efficacious with 95% confidence interval between −10.5% and 49.3%), but these data are yet to be published in a peer-reviewed journal [55].

Though Imbokodo was completed, Mosaico, the similar trial, is not yet finished and has some differences in vaccine components and patient population [53]. This vaccine has gp140 immunogens from different HIV strains, rather than clade C specific as in Imbokodo [6, 44]. Mosaico enrolled gay men and other men who have sex with men as well as transgender women. This study is still ongoing in hopes that the slight differences between immunogens and transmission mode will result in some vaccine efficacy [55, 56].
