Development and testing of nucleic acid-based vaccine for HIV and other indications

The development of a safe and effective vaccine to prevent HIV infection and to improve treatment strategies aiming to reduce/eliminate the virus reservoir are currently at the forefront of our research. We developed the key methodology to express HIV antigens at high level from RNA/codon-optimized genes, which allows efficient antigen production when expressed from simple DNA plasmids or as part of recombinant viral vectors. Immunogenicity is augmented in the presence of cytokines, i.e., IL-12 DNA co-administration. We optimized DNA delivery with the result of achieving systemic and mucosal immune responses. Using DNA+Protein combination vaccines, the magnitude, breadth and longevity of the immune responses increased resulting in significant improved protection from infection in the SIV/SHIV NHP model (Patel, PNAS 110: 2975, 2013; Jalah, PLoS One 9: e91550, 2014, Felber, Cell Reports, 31:107624, 2020). The vaccine platform combines the delivery of both vaccine components into the same anatomical site targeting the same draining lymph node. We recently reported that co-administration in the same site showed a 67% reduction in per exposure acquisition risk relative to the controls, whereas neither animals vaccinated with DNA and protein in separate sites, nor the controls were protected from an intravaginal SHIV CH505 virus challenge (Felber, Cell Reports, 31:107624, 2020). These data have important implications for other vaccine modalities because combination vaccines are typically administered in separate anatomical sites. We hypothesize that optimization of immunogens to better target the rare B cell precursor, combined with the co-administration of vaccine vector and protein in same draining lymph nodes could provide an immunological advantage over current protocols, resulting in significantly improved protection and we are exploring this mechanism in the macaque model.

We applied our experience in developing HIV vaccines towards SARS-CoV-2 (Rosati, PLoS Pathog 17:e1009701; 2021). The different Spike DNA-based vaccine regimens induced robust antibody and T cell responses able to effectively mediate protection and to control SARS-CoV-2 infection in macaques. Importantly, a vaccine regimen comprising simultaneous co-immunization of DNA and protein at the same anatomical site showed was more effective than DNA alone in inducing protective immune responses and controlling SARS-CoV-2 infection. We are now exploring novel DNA delivery methods to achieve better immunity of DNA-only vaccine to simplify the vaccine platform.

We developed novel molecules to direct and focus humoral immune responses to V2 domain of the HIV gp120 Env. We reported that priming with this DNA altered the hierarchy of humoral immune responses to V2 region epitopes, providing a method for more efficient induction and maintenance of V2-specific Env Abs associated with reduced risk of HIV infection (Devasundaram, J Virol 95:e01193, 2020). We are testing the hypothesis whether these responses translate to better protection in the macaque model.

Development and clinical testing of Conserved Element (CE) vaccine

Considering the diversity of the different HIV clades, we are focusing on highly conserved regions of HIV to induce immune responses to nearly invariable proteome segments, essential for the function of the virus, while excluding responses to variable and potentially immunodominant "decoy" epitopes (Kulkarni, PLoS One 9: e86254, 2014; Kulkarni, PLoS One 9: e111085, 2014; Hu, Hum. Vaccin. Immunother. 14: 2163, 2018). The method of priming with CE DNA and boosting with CE+gag DNA maximizes both magnitude and breadth (Kulkarni, PLoS One 9: e86254. 2014; Kulkarni, PLoS One 9: e111085, 2014; Hu, J. Immunol. 197: 3999, 2016). Thus, we identified a novel and effective strategy to maximize responses against Gag and provide a novel strategy to shift the immunodominance hierarchy and to induce robust immune responses to subdominant epitopes, effectively targeting the Achilles' heel of the virus.

We have translated the HIV CE DNA vaccine concept to the clinic in the HIV Vaccine Trial Network (HVTN)/DAIDS/NIAID-supported clinical trial (HVTN 119; NCT03181789), in a phase I/IIb trial (ACTG A5369; NCT03560258) supported by the AIDS Clinical Trial Group,  in HIV-infected persons under HAART, and trial NCT04357821 supported by amfAR and UCSF (open Q4, 2020) as part of a combinatorial therapy to induce an HIV remission. HVTN 119 and A5369 combine several of milestones (DNA expression vectors, adjuvants, and delivery) we have achieved over many years in vaccine development. Together, these trials will allow us to explore the translation of our data from mice and macaques to humans and initial data analysis supports our achievement of this goal. These two trials offer another unique opportunity to directly compare the same vaccines in two different cohorts, naïve and HIV-infected on ART, using the same methodology, and will provide novel insight in the development in immune responses comparing the two cohorts.

We have further expanded the CE vaccine regimen in collaboration with CureVac, Inc. as an mRNA/lipid nanoparticle (LNP) formulation in the macaque model (Valentin, Frontiers Immunol, 2022, in press). The mRNA/LNP formulation induced robust, durable antibody responses but low adaptive T-cell responses, a feature also shared with the current COVID-19 mRNA vaccine. On the other hand, the mRNA/LNP vaccine was able to strongly boost pre-existing cellular response suggesting that it could be useful in heterologous prime/boost modality and in immune therapeutic interventions against HIV infection or other chronic human diseases. Our studies have provided useful information about different nucleic acid vaccine platforms and guide further clinical development.

COVID-19 vaccine in naïve individuals and cancer patients

We have been studying the immune response of COVID-19 cohorts longitudinally to characterize the nature and longevity of immune response (Rosati M, Am J Hematol: 97:E3, 2022; Rosati Frontiers Immunol 12: 793953, 2021; Thomopoulos, Viruses 1:1844; 2021; Terpos, Eur J Intern Med, 89:87,2021; Pappa, Microorganisms 9, 806, 2021; Terpos, Microorganisms. 8:1885, 2020). The analysis of natural infection is providing important information for the design of vaccine strategies. To characterize adaptive and innate immune responses in SARS-CoV2 vaccinated persons, we identified early responses to vaccination that are important in shaping both humoral and cellular protective immunity (Bergamaschi, Cell Rep 36:109504; 2021; Bergamaschi, Frontiers Immunol 3:899972, 2022). We characterized the cytokine and chemokine responses to BNT162b2 mRNA (Pfizer/BioNtech) vaccinations in antigen-naïve and in previously COVID-19-infected individuals and in patients with hematological malignancies (NCT04743388). We identified a systemic signature including IL-15, IFN-gamma, IP-10/CXCL10, TNF-alpha and IL-6. Transient increases in IL-15 and IFN-gamma levels early after boost correlated with Spike antibody levels, supporting their use as biomarkers of effective humoral immunity development in response to vaccination. We are expanding our research of the development of adaptive and innate immune responses upon COVID-19 vaccination to other cancer cohorts.

hetIL-15 in cancer immunotherapy and HIV cure

Understanding of the cellular mechanisms controlling expression shed new light in the biology and regulation of IL-15, led to the identification of the bioactive heterodimeric form of IL-15 and provided methods for the efficient production and clinical application of this cytokine (Bergamaschi, J. Biol. Chem. 283: 4189, 2008; Bergamaschi, Blood 120: e1-8, 2012; Bergamaschi, Cancers 13, 2021). We have shown that hetIL-15 greatly increases lymphocyte infiltration in several tumors in mouse models and in macaques, suggesting a general method to increase lymphocyte infiltration, which is associated with anti-tumor activity.  We have performed first-in-human clinical trials of hetIL-15 in metastatic cancers (Conlon, JITC:e003388. 2021) and also in combination with anti-PD-1 check point inhibitor (NCT02452268; collaboration with Novartis).

hetIL-15 is extensively studied in mouse tumor models where we demonstrated the rapid interaction of lymphoid and myeloid cell networks resulting in changes in numbers and migration of different cell populations. Extensive transcriptomics and proteomics analysis has revealed additional pathways affected by hetIL-15 (Bergamaschi, J. Immunother. Cancer: 8:e000599, 2020; Bergamaschi, Cancers 13, 2021). We have studied the effects of hetIL-15 in the number and properties of Dendritic Cells (DC) in tumors which increase upon hetIL-15 treatment. DC participate in a network of cells that are induced during hetIL-15 treatment and in turn support more recruitment of effector cells in tumor sites. Studies in mouse orthotopic models show that DC populations correlate with tumor regression and reveal additional beneficial effects of hetIL-15 in inducing or enhancing long term protective immune response against tumors. Thus, hetIL-15, a cytokine directly affecting lymphocytes and inducing cytotoxic cells, has also an indirect rapid and significant effect on the recruitment of myeloid cells, initiating a cascade for tumor elimination though innate and adoptive immune mechanisms.

In addition to cancer immunotherapy, IL-15 has generated strong interest for clinical use to treat HIV infection, especially in protocols targeting viral eradication or a functional cure. The use of IL-15 as an immune therapeutic agent against HIV infection is based on its effects as a growth factor and key regulator of cytotoxic responses mediated by both the innate (NK cells) and the adaptive (CTL) arms of the immune system. Importantly, hetIL-15 treatment promotes the entrance of cytotoxic (GrzB+) CD8+ T cells in the B cell follicles, areas within the LN where CTL are typically excluded and where SIV/HIV infected follicular helper CD4+ T cells reside. hetIL-15 treatment led to significant decrease in cell-associated viral RNA within the LN as well as in plasma viremia in SHIV infected macaques (Watson, PLoS Pathog. 14: e1006902, 2018). In collaborative effort, we further reported that a IL-15 transcriptional signature response to a viral RhCMV/SIV vaccine strongly correlated with protection (Barrenas, PLoS Pathog 17:e1009278; 2021). We have expanded this concept and are testing the contribution of a combinatorial treatment including hetIL-15 in reducing reservoir in the SIV infected ART-treated macaques.