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Purpose: Adoptive cell transfer (ACT) is a promising immunotherapeutic approach for cancer. Host lymphodepletion is associated with favorable ACT therapy outcomes, but it may cause detrimental effects in humans. We tested the hypothesis that IL15 administration enhances ACT in the absence of lymphodepletion. We previously showed that bioactive IL15 in vivo comprises a stable complex of the IL15 chain with the IL15 receptor alpha chain (IL15Rα), termed heterodimeric IL15 (hetIL15).Experimental Design: We evaluated the effects of the combination regimen ACT + hetIL15 in the absence of lymphodepletion by transferring melanoma-specific Pmel-1 T cells into B16 melanoma-bearing mice.Results: hetIL15 treatment delayed tumor growth by promoting infiltration and persistence of both adoptively transferred Pmel-1 cells and endogenous CD8+ T cells into the tumor. In contrast, persistence of Pmel-1 cells was severely reduced following irradiation in comparison with mice treated with hetIL15. Importantly, we found that hetIL15 treatment led to the preferential enrichment of Pmel-1 cells in B16 tumor sites in an antigen-dependent manner. Upon hetIL15 administration, tumor-infiltrating Pmel-1 cells showed a “nonexhausted” effector phenotype, characterized by increased IFNγ secretion, proliferation, and cytotoxic potential and low level of PD-1. hetIL15 treatment also resulted in an improved ratio of Pmel-1 to Treg in the tumor.Conclusions: hetIL15 administration improves the outcome of ACT in lymphoreplete hosts, a finding with significant implications for improving cell-based cancer immunotherapy strategies. Clin Cancer Res; 23(11); 2817–30. ©2016 AACR.

Supplementary Figure 1. IL-15 availability promotes proliferation and maintenance of transferred CD8+ T in the spleen of recipient mice; Supplementary Figure 2. Gating strategy for the identification of adoptively transferred Pmel-1 cells and endogenous CD8+ T cells infiltrating the tumor; Supplementary Figure 3. Absolute counts of splenic Pmel-1 and CD8+ T cells are profoundly affected by hetIL-15 treatment; Supplementary Figure 4. Evaluation of PD-1, Ki67 and GzmB expression by tumorinfiltrating Pmel-1 cells; Supplementary Figure 5. Evaluation of PD-1, Ki67 and GzmB expression by tumorinfiltrating CD8+ T cells; Supplementary Table 1. Hematological parameters during ACT treatments.

There is an urgent need for a malaria vaccine that can prevent severe disease in young children and adults. Despite earlier work showing an immunological mechanism for preventing infection and reducing disease severity, there is currently no reliable vaccine that can provide durable protection. In part, this may reflect a limited number of ways that the host can respond to the NANP repeat sequences of circumsporozoite protein (CSP) in the parasite. In addition, it may reflect antigenic escape by the parasite from protective antibodies. To be successful, a vaccine must protect against repeated exposure to infected mosquitoes in endemic areas. We have created a series of live viral vectors based on the rubella vaccine strain that express multiple tandem repeats of NANP, and we demonstrate immunogenicity in a rhesus macaque model. We tested the vectors in a sequential immunization strategy. In the first step, the animals were primed with CSP-DNA vaccine and boosted with rubella/CSP vectors. In the second step, we gave rubella/CSP vectors again, followed by recombinant CSP protein. Following the second step, antibody titers were comparable to adult exposure to malaria in an endemic area. The antibodies were specific for native CSP protein on sporozoites, and they persisted for at least 1½ years in two out of three macaques. Given the safety profile of rubella vaccine in children, these vectors could be most useful in protecting young children, who are at greatest risk of severe malarial disease.

Introduction: Metabolic fitness and T cell survival are crucial in anti-tumor responses because nutrients are often scarce and other regulatory molecules may be unfavorable in the tumor microenvironment leading to T cell dysfunction, stress, and apoptosis. Tumor-infiltrating cytotoxic CD8+T cells frequently acquire an altered state of differentiation referred to as “exhaustion” and, as a result, they fail to control tumor outgrowth. IL-15 cytokine stimulates the generation, proliferation and cytotoxic function of tumor specific CD8+ T cells and NK cells. The objective of this study was to assess the effects of hetIL-15 immunotherapy in triple negative breast cancer tumors (TNBC), to evaluate the metabolic profile of the tumor-infiltrating T cells and to study any potential synergy using a Fatty Acid Metabolism Modulator (FAMM) to enhance T cell metabolism. Study design and methods: We used the murine EO771 orthotopic breast cancer model to study the efficacy of locoregional administration of hetIL-15 immunotherapy in combination with a FAMM. We monitored the effect of treatment on number, metabolism and mitochondrial function of the tumor-infiltrating immune cells by flow cytometry, Seahorse flux analysis and Mitotracker, 2-NBDG and/or Bodipy staining. Results: hetIL-15 locoregional administration, as as single agent, resulted in complete regression in 40% of the treated animals and increased survival. Tumor-infiltrating cytotoxic CD8+T and NK cells increased in hetIL-15 treated tumors and showed enhanced activation and proliferation. Metabolic flux analysis of the tumor-infiltrating cytotoxic CD8+T cells from hetIL-15-treated mice confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity, which supports an activated/non exhausted phenotype of these hetIL-15 treated effector cells. Since, further promoting fatty acid (FA) catabolism improves the tumor-infiltrated CD8+T cells’s ability to slow tumor progression, we combined hetIL-15 immunotherapy with a FAMM. Combination therapy resulted in increased mitochondrial function, FA uptake and OCR, revealing a more metabolically active phenotype compared to the tumor-infiltrating CD8+T cells from hetIL-15 monotherapy group. In addition, combined treatment of IL-15 immunotherapy and FAMM resulted in statistically significant EO771 tumor growth delay and complete eradication of the tumors in 85% of mice. Conclusions: Our results indicate that hetIL-15 synergizes with metabolic reprogramming of T cells to achieve superior antitumor efficacy and complete cures. We suggest that metabolic reprogramming of tumor-specific CD8+Tcells might represent a strategy to promote survival in the metabolically hostile TME as part of an approach to enhance the clinical efficacy of immunotherapy. Citation Format: Sevasti Karaliota, Dimitris Stellas, Vasiliki Stravokefalou, Breana Myers, Barbara K. Felber, George N. Pavlakis. Heterodimeric IL-15 (hetIL-15) immunotherapy synergizes with Fatty Acid Metabolism Modulator (FAMM) to eradicate TNBC EO771 murine tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5082.

Durability of SARS-CoV-2 Spike antibody responses after infection provides information relevant to understanding protection against COVID-19 in humans. We report the results of a sequential evaluation of anti-SARS-CoV-2 antibodies in convalescent patients with a median follow-up of 14 months (range 12.4-15.4) post first symptom onset. We report persistence of antibodies for all four specificities tested [Spike, Spike Receptor Binding Domain (Spike-RBD), Nucleocapsid, Nucleocapsid RNA Binding Domain (N-RBD)]. Anti-Spike antibodies persist better than anti-Nucleocapsid antibodies. The durability analysis supports a bi-phasic antibody decay with longer half-lives of antibodies after 6 months and antibody persistence for up to 14 months. Patients infected with the Wuhan (WA1) strain maintained strong cross-reactive recognition of Alpha and Delta Spike-RBD but significantly reduced binding to Beta and Mu Spike-RBD. Sixty percent of convalescent patients with detectable WA1-specific NAb also showed strong neutralization of the Delta variant, the prevalent strain of the present pandemic. These data show that convalescent patients maintain functional antibody responses for more than one year after infection, suggesting a strong long-lasting response after symptomatic disease that may offer a prolonged protection against re-infection. One patient from this cohort showed strong increase of both Spike and Nucleocapsid antibodies at 14 months post-infection indicating SARS-CoV-2 re-exposure. These antibodies showed stronger cross-reactivity to a panel of Spike-RBD including Beta, Delta and Mu and neutralization of a panel of Spike variants including Beta and Gamma. This patient provides an example of strong anti-Spike recall immunity able to control infection at an asymptomatic level. Together, the antibodies from SARS-CoV-2 convalescent patients persist over 14 months and continue to maintain cross-reactivity to the current variants of concern and show strong functional properties.

Patients with symptomatic monoclonal gammopathies have impaired humoral responses to COVID-19 vaccination. Their ability to recognize SARS-CoV-2 Omicron variants is of concern. We compared the response to BNT162b2 mRNA vaccinations of patients with multiple myeloma (MM, n = 60) or Waldenstrom’s macroglobulinemia (WM, n = 20) with healthy vaccine recipients (n = 37). Patient cohorts on active therapy affecting B cell development had impaired binding and neutralizing antibody (NAb) response rate and magnitude, including several patients lacking responses, even after a 3rd vaccine dose, whereas non-B cell depleting therapies had a lesser effect. In contrast, MM and WM cohorts off-therapy showed increased NAb with a broad response range. ELISA Spike-Receptor Binding Domain (RBD) Ab titers in healthy vaccine recipients and patient cohorts were good predictors of the ability to neutralize not only the original WA1 but also the most divergent Omicron variants BA.4/5. Compared to WA1, significantly lower NAb responses to BA.4/5 were found in all patient cohorts on-therapy. In contrast, the MM and WM cohorts off-therapy showed a higher probability to neutralize BA.4/5 after the 3rd vaccination. Overall, the boost in NAb after the 3rd dose suggests that repeat vaccination of MM and WM patients is beneficial even under active therapy.

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with strain 68–1 Rhesus Cytomegalovirus (RhCMV) vectors expressing SIV proteins (RhCMV/SIV) results in a binary outcome: stringent control and subsequent clearance of highly pathogenic SIV in ~55% of vaccinated RMs with no protection in the remaining 45%. Although previous work indicates that unconventionally restricted, SIV-specific, effector-memory (EM)-biased CD8+ T cell responses are necessary for efficacy, the magnitude of these responses does not predict efficacy, and the basis of protection vs. non-protection in 68–1 RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that 68–1 RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including immune cell, toll-like receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly correlating with subsequent vaccine efficacy. Treatment of a separate RM cohort with IL-15 confirmed the central involvement of this cytokine in the protection signature, linking the major innate and adaptive immune gene expression networks that correlate with RhCMV/SIV vaccine efficacy. This change-from-baseline IL-15 response signature was also demonstrated to significantly correlate with vaccine efficacy in an independent validation cohort of vaccinated and challenged RMs. The differential IL-15 gene set response to vaccination strongly correlated with the pre-vaccination activity of this pathway, with reduced baseline expression of IL-15 response genes significantly correlating with higher vaccine-induced induction of IL-15 signaling and subsequent vaccine protection, suggesting that a robust de novo vaccine-induced IL-15 signaling response is needed to program vaccine efficacy. Thus, the RhCMV/SIV vaccine imparts a coordinated and persistent induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ T cell function, that support the ability of vaccine-elicited unconventionally restricted CD8+ T cells to mediate protection against SIV challenge., Author summary SIV insert-expressing vaccine vectors based on strain 68–1 RhCMV elicit robust, highly effector-memory-biased, unconventionally restricted T cell responses that are associated with an unprecedented level of SIV control after challenge (replication arrest leading to clearance) in slightly over half of vaccinated monkeys. Since efficacy among monkeys vaccinated with the effective 68–1 vaccine is not predicted by standard measures of immunogenicity, we used functional genomics analysis of RhCMV/SIV vaccinated monkeys with known challenge outcomes to identify immune correlates of protection. We found that vaccine efficacy significantly correlates with a vaccine-induced response to IL-15 that includes modulation of immune cell, inflammation, TLR signaling, and cell death programming response pathways. These data suggest that RhCMV/SIV efficacy results from a coordinated and sustained vaccine-mediated induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ effector-memory T cell function, that cooperates with vaccine-elicited CD8+ T cells to mediate efficacy.

Human interleukin-12 (hIL-12) is a heparin-binding cytokine whose activity was previously shown to be enhanced by heparin and other sulfated glycosaminoglycans. The current study investigated the mechanisms by which heparin increases hIL-12 activity. Using multiple human cell types, including natural killer cells, an IL-12 indicator cell line, and primary peripheral blood mononuclear and T cells, along with bioactivity, flow cytometry, and isothermal titration calorimetry assays, we found that heparin-dependent modulation of hIL-12 function correlates with several of heparin's biophysical characteristics, including chain length, sulfation level, and concentration. Specifically, only heparin molecules longer than eight saccharide units enhanced hIL-12 activity. Furthermore, heparin molecules with three sulfate groups per disaccharide unit outperformed heparin molecules with one or two sulfate groups per disaccharide unit in terms of enhanced hIL-12 binding and activity. Heparin also significantly reduced the EC(50) value of hIL-12 by up to 11.8-fold, depending on the responding cell type. Cytokine-profiling analyses revealed that heparin affected the level, but not the type, of cytokines produced by lymphocytes in response to hIL-12. Interestingly, although murine IL-12 also binds heparin, heparin did not enhance its activity. Using the gathered data, we propose a model of hIL-12 stabilization in which heparin serves as a co-receptor enhancing the interaction between heterodimeric hIL-12 and its receptor subunits. The results of this study provide a foundation for further investigation of heparin's interactions with IL-12 family cytokines and for the use of heparin as an immunomodulatory agent.

The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized Wuhan-Hu-1 SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The antibodies recognized and potently neutralized a panel of different Spike variants including Alpha, Delta, Epsilon, Eta and A.23.1, but to a lesser extent Beta and Gamma. The DNA-only vaccine regimens were compared to a regimen that included co-immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques., Author summary Anti-Spike neutralizing antibodies provide strong protection against SARS-CoV-2 infection in animal models, and correlate with protection in humans, supporting the notion that induction of strong humoral immunity is key to protection. We show induction of robust antibody and T cell responses by different Spike DNA-based vaccine regimens able to effectively mediate protection and to control SARS-CoV-2 infection in the rhesus macaque model. This study provides the opportunity to compare vaccines able to induce different humoral and cellular immune responses in an effort to develop durable immunity against the SARS-CoV-2. A vaccine regimen comprising simultaneous co-immunization of DNA and Protein at the same anatomical site showed best neutralizing abilities and was more effective than DNA alone in inducing protective immune responses and controlling SARS-CoV-2 infection. Thus, an expansion of the DNA vaccine regimen to include co-immunization with Spike protein may be of advantage also for SARS-CoV-2.

Elucidating the characteristics of human immune response against SARS-CoV-2 is of high priority and relevant for determining vaccine strategies. We report the results of a follow-up evaluation of anti-SARS-CoV-2 antibodies in 148 convalescent plasma donors who participated in a phase 2 study at a median of 8.3 months (range 6.8-10.5 months) post first symptom onset. Monitoring responses over time, we found contraction of antibody responses for all four antigens tested, with Spike antibodies showing higher persistence than Nucleocapsid antibodies. A piecewise linear random-effects multivariate regression analysis showed a bi-phasic antibody decay with a more pronounced decrease during the first 6 months post symptoms onset by analysis of two intervals. Interestingly, antibodies to Spike showed better longevity whereas their neutralization ability contracted faster. As a result, neutralizing antibodies were detected in only 76% of patients at the last time point. In a multivariate analysis, older age and hospitalization were independently associated with higher Spike, Spike-RBD, Nucleocapsid, N-RBD antibodies and neutralizing antibody levels. Results on persistence and neutralizing ability of anti-SARS-CoV-2 antibodies, especially against Spike and Spike-RBD, should be considered in the design of future vaccination strategies.

Simple Summary The rapidly expanding field of cancer immunotherapy uses diverse technologies, including cytokines, T cells, and antibody administration, with the aim to induce effective immune responses leading to tumor control. Interleukin-15 (IL-15), a cytokine discovered in 1994, supports the homeostasis of cytotoxic immune cells and shows promise as an anti-tumor agent. Many studies have elucidated IL-15 synthesis, regulation and biological function and explored its therapeutic efficacy in preclinical cancer models. Escherichia coli-derived single-chain IL-15 was tested in the first in-human trial in cancer patients. Its effects were limited by the biology of IL-15, which in vivo comprises a complex of the IL-15 chain with the IL-15 receptor alpha (IL-15Rα) chain, together forming the IL-15 heterodimer (hetIL-15). Currently, single-chain IL-15 and several heterodimeric IL-15:IL-15Rα variants (hetIL-15, N-803 and RLI) are being tested in clinical trials. This review presents a summary of contemporary preclinical and clinical research on IL-15. Abstract Immunotherapy has emerged as a valuable strategy for the treatment of many cancer types. Interleukin-15 (IL-15) promotes the growth and function of cytotoxic CD8+ T and natural killer (NK) cells. It also enhances leukocyte trafficking and stimulates tumor-infiltrating lymphocytes expansion and activity. Bioactive IL-15 is produced in the body as a heterodimeric cytokine, comprising the IL-15 and the so-called IL-15 receptor alpha chain that are together termed “heterodimeric IL-15” (hetIL-15). hetIL-15, closely resembling the natural form of the cytokine produced in vivo, and IL-15:IL-15Rα complex variants, such as hetIL-15Fc, N-803 and RLI, are the currently available IL-15 agents. These molecules have showed favorable pharmacokinetics and biological function in vivo in comparison to single-chain recombinant IL-15. Preclinical animal studies have supported their anti-tumor activity, suggesting IL-15 as a general method to convert “cold” tumors into “hot”, by promoting tumor lymphocyte infiltration. In clinical trials, IL-15-based therapies are overall well-tolerated and result in the expansion and activation of NK and memory CD8+ T cells. Combinations with other immunotherapies are being investigated to improve the anti-tumor efficacy of IL-15 agents in the clinic.

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with Rhesus Cytomegalovirus (RhCMV) vectors expressing SIV proteins (RhCMV/SIV) results in a binary outcome: stringent control and subsequent clearance of highly pathogenic SIV in ~55% of vaccinated RMs with no protection in the remaining 45%. Although previous work suggests that unconventionally restricted, SIV-specific, effector-memory (EM)-biased CD8+ T cell responses are necessary for efficacy, the magnitude of these responses does not predict efficacy, and the basis of protection vs. non-protection in RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including non-canonical T cell receptor (TCR), toll-lie receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly predicting protection. The IL-15 gene expression signature was further evaluated in an independent RM IL-15 treatment cohort, revealing that in whole blood the response to IL-15 is inclusive of innate and adaptive immune gene expression networks that link with RhCMV/SIV vaccine efficacy. We also show that this IL-15 response signature similarly tracks with vaccine protection in an independent RhCMV/SIV vaccination/SIV challenge RM validation cohort. Thus, the RhCMV/SIV vaccine imparts a coordinated and persistent induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ T cell function, that enable vaccine-elicited CD8+ T cells to mediate protection against highly pathogenic SIV challenge.Author SummarySIV insert-expressing vaccine vectors based on strain 68-1 RhCMV elicit robust, highly effector-memory-biased T cell responses that are associated with an unprecedented level of SIV control after challenge (replication arrest leading to clearance) in slightly over half of vaccinated monkeys. Since efficacy is not predicted by standard measures of immunogenicity, we used functional genomics analysis of RhCMV/SIV vaccinated monkeys with known challenge outcomes to identify immune correlates of protection. We found that arrest of viral replication after challenge significantly correlates with a vaccine-induced response to IL-15 that includes modulation of T cell, inflammation, TLR signaling, and cell death programming. These data suggest that RhCMV/SIV efficacy is not based on chance, but rather, results from a coordinated and sustained vaccine-mediated induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ effector-memory T cell function, that enable vaccine-elicited CD8+ T cells to mediate efficacy.

Early responses to vaccination are important in shaping both humoral and cellular protective immunity. Dissecting innate vaccine signatures may predict immunogenicity and help optimizing efficacy of mRNA and other vaccine strategies. We characterized the cytokine and chemokine responses to the 1st and 2nd dose of the BNT162b2 mRNA (Pfizer/BioNtech) vaccine in antigen-naive vaccine recipients and individuals previously infected by COVID-19 (NCT04743388). Transient increases in IL-15 and IFN-γ levels early after boost correlated with Spike-RBD antibody levels, supporting their possible use as biomarkers of successful vaccination. We identified a systemic signature including IL-15, IFN-γ and IP-10/CXCL10 increase after the 1st vaccination, which was enriched by TNF-α and IL-6 after the 2nd vaccination. In vaccine recipients with history of COVID-19 infection, a single vaccine dose resulted in both strong cytokine induction and antibody titers similar to the ones observed upon booster vaccination in antigen-naive individuals, a result with potential implication for future public health recommendations. Funding Information: Funding: This research was supported [in part] by the Intramural Research Program of the NIH, NCI to G.N.P. and B.K.F. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All study procedures were carried out in accordance with the declaration of Helsinki (18th World Medical Association Assembly), its subsequent amendments, the Greek regulations and guidelines, as well as the good clinical practice guidelines (GCP) as defined by the International Conference of Harmonization. The study was also approved by the local ethic committee of Alexandra General Hospital (no 15/23 December 2020).

Introduction: Metabolic fitness and T cell survival are crucial in anti-tumor responses because nutrients are often scarce and other regulatory molecules may be unfavorable in the tumor microenvironment leading to T cell dysfunction, stress, and apoptosis. Tumor-infiltrating cytotoxic CD8+T cells often acquire an altered state of differentiation referred to as “exhaustion” and, as a result, they fail to control tumor outgrowth. IL-15 cytokine stimulates the generation, the proliferation and the cytotoxic function of tumor specific CD8+ T cells and NK cells. The objective of this study was to assess the effects of hetIL-15 immunotherapy after locoregional administration in triple negative breast cancer tumors (TNBC) and to evaluate the metabolic profile of the tumor infiltrating T cells. Study design and methods: We studied the therapeutic efficacy of hetIL-15 immunotherapy in murine EO771 orthotopic breast cancer model. We monitored the effect of the treatment on tumor size and immune infiltration using flow cytometry and immunohistochemistry. We also evaluated hetIL-15 effects on the cell metabolism and the mitochondrial function of the tumor-infiltrating immune cells, using flow cytometry, Seahorse flux analysis, Mitotracker, 2-NBDG and/or Bodipy staining. Results: hetIL-15 peritumoral administration, as monotherapy resulted in complete regression in 40% of the treated animals and increased survival. We demonstrated that tumor infiltrating cytotoxic CD8+T and NK cells were increased in hetIL-15 treated tumors and showed enhanced activation and proliferation, resulting in enhanced intratumoral immunological killing. Metabolic flux analysis of the tumor-infiltrating CD8+T cells from treated mice confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity, which supports an activated/non exhausted phenotype of these hetIL-15 treated effector cells. Consistent with the above finding, tumor infiltrated CD8+T cells from hetIL-15 treated mice showed increased mitochondrial potential and fatty acid uptake, as evidenced by increased Mitotracker and Bodipy staining, respectively. In addition, tumor infiltrating CD8+T cells from hetIL-15 treated mice presented elevated extracellular acidification rate (ECAR) and showed a pronounced shift in the OCR to ECAR ratio in comparison to control, confirming their increased proliferating status. Conclusions: Our results indicate that hetIL-15 not only increases the infiltration and the cytotoxic properties of the CD8+T cells inside the tumors, but also enhances the metabolic reprogramming of T cells to achieve superior antitumor efficacy. We suggest that metabolic reprogramming of tumor-specific CD8+T cells might represent a strategy to promote survival in the metabolically hostile TME as part of an approach to enhance the clinical efficacy of immunotherapy. Citation Format: Dimitris Stellas, Sevasti Karaliota, Vasiliki Stravokefalou, Breana Myers, Barbara K. Felber, George N. Pavlakis. Heterodimeric IL-15 (hetIL-15) immunotherapy reverses CD8+T cell metabolic dysfunction in murine breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5603.

Introduction: Metastasis is responsible for most of the cancer-related deaths (~ 90%). Metastatic triple negative breast cancer (TNBC) has poor survival and chemotherapy pre- and post-surgery has been the standard treatment for decades. Polymorphonuclear - myeloid derived suppressor cells (PMN-MDSCs) are implicated in the process of metastasis. IL-15 stimulates the proliferation and cytolytic activity of CD8+T cells and Natural Killer cells (NKs) and has been reported to have anti-metastatic activity. We have produced the native heterodimeric form of IL-15 (hetIL-15) which has advanced in clinical trials due to its anticancer activities. Here, we report the anti-metastatic effects of hetIL-15 alone and in combination with doxorubicin and surgery using the 4T1 mouse model of TNBC. Study design and methods: We studied the efficacy of the chemo-immunotherapeutic regimen with surgery in a neoadjuvant and adjuvant setting or without surgery. The animals were evaluated regarding metastatic disease and survival. The anti-metastatic effect was evaluated by examining the metastatic foci in lungs and the circulating tumor cells (CTCs) in blood by histology and clonogenic assays. Treatment effects in immune populations in blood, spleen and lungs were evaluated by flow cytometry analysis and immunohistochemistry (IHC). Results: Mice treated with hetIL-15 in combination with doxorubicin had significantly better survival in comparison to monotherapy treatments and control group. H&E histological analysis in the lungs revealed substantially fewer metastatic foci after hetIL-15 monotherapy, and even fewer in the combination group. Furthermore, clonogenic assays from lungs and blood, reinforced these findings showing significantly lower numbers of tumor colonies upon hetIL-15 monotherapy and combination treatment. Immune profiling evaluation of blood, spleen and lungs by flow cytometry and IHC revealed a significant systemic increase of cytotoxic effector cells (CD8+T cells and NKs), combined with reduction of immunosuppressive populations (PMN-MDSCs) especially in the combination group. These findings suggest a synergistic effect of doxorubicin and hetIL-15 in controlling the metastatic disease. Lastly, hetIL-15 monotherapy or co-administration with doxorubicin, together with surgery, led to the cure of approximately 50% of the treated mice. Conclusions: Our results demonstrate that hetIL-15 reduces metastatic disease and synergizes with doxorubicin and surgery to maximize the effectiveness of the treatment against breast cancer in mice. We suggest that the effect of hetIL-15 on metastatic disease is both at the level of dissemination and also colonization in the metastatic sites. This should be further explored in the clinical setting as a neoadjuvant and adjuvant therapy in combination with chemotherapy and surgery for the treatment of TNBC. Citation Format: Vasiliki Stravokefalou, Dimitris Stellas, Sevasti Karaliota, Breana Myers, Cristina Bergamaschi, Konstantinos Dimas, Barbara K. Felber, George N. Pavlakis. hetIL-15 decreases tumor cell dissemination and colonization and synergizes with chemotherapy and surgery to cure murine 4T1 breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2441.

The aim of this work was to design and test a vaccine regimen focusing the immune response on targets associated with infection prevention. We demonstrated that priming with a DNA vaccine expressing only the HIV Env V1V2 region induces Ab responses targeting the critical region in V2 associated with protection. This work shows that V1V2 scaffold DNA priming immunization provides a method to focus immune responses to the desired target region, in the absence of immune interference by other epitopes. This induced immune responses with improved recognition of epitopes important for protective immunity, namely, V2-specific humoral immune responses inversely correlating with HIV risk of infection in the RV144 trial., The RV144 vaccine trial revealed a correlation between reduced risk of HIV infection and the level of nonneutralizing-antibody (Ab) responses targeting specific epitopes in the second variable domain (V2) of the HIV gp120 envelope (Env) protein, suggesting this region as a target for vaccine development. To favor induction of V2-specific Abs, we developed a vaccine regimen that included priming with DNA expressing an HIV V1V2 trimeric scaffold immunogen followed by booster immunizations with a combination of DNA and protein in rhesus macaques. Priming vaccination with DNA expressing the HIV recombinant subtype CRF01_AE V1V2 scaffold induced higher and broader V2-specific Ab responses than vaccination with DNA expressing CRF01_AE gp145 Env. Abs recognizing the V2 peptide that was reported as a critical target in RV144 developed only after the priming immunization with V1V2 DNA. The V2-specific Abs showed several nonneutralizing Fc-mediated functions, including ADCP and C1q binding. Importantly, robust V2-specific Abs were maintained upon boosting with gp145 DNA and gp120 protein coimmunization. In conclusion, priming with DNA expressing the trimeric V1V2 scaffold alters 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. IMPORTANCE The aim of this work was to design and test a vaccine regimen focusing the immune response on targets associated with infection prevention. We demonstrated that priming with a DNA vaccine expressing only the HIV Env V1V2 region induces Ab responses targeting the critical region in V2 associated with protection. This work shows that V1V2 scaffold DNA priming immunization provides a method to focus immune responses to the desired target region, in the absence of immune interference by other epitopes. This induced immune responses with improved recognition of epitopes important for protective immunity, namely, V2-specific humoral immune responses inversely correlating with HIV risk of infection in the RV144 trial.

An efficacious human immunodeficiency virus (HIV) vaccine will likely require induction of both mucosal and systemic immune responses. We compared the immunogenicity and protective efficacy of two mucosal/systemic vaccine regimens and investigated their effects on the rectal microbiome. Rhesus macaques were primed twice mucosally with replication-competent adenovirus type 5 host range mutant (Ad5hr)-simian immunodeficiency virus (SIV) recombinants and boosted twice intramuscularly with ALVAC-SIV recombinant plus SIV gp120 protein or with DNA for SIV genes and rhesus interleukin-12 plus SIV gp120 protein. Controls received empty Ad5hr vector and alum adjuvant only. Both regimens elicited strong, comparable mucosal and systemic cellular and humoral immunity. Prevaccination rectal microbiomes of males and females differed and significantly changed over the course of immunization, most strongly in females after Ad5hr immunizations. Following repeated low-dose intrarectal SIV challenges, both vaccine groups exhibited modestly but significantly reduced acute viremia. Male and female controls exhibited similar acute viral loads; however, vaccinated females, but not males, exhibited lower levels of acute viremia, compared to same-sex controls. Few differences in adaptive immune responses were observed between the sexes. Striking differences in correlations of the rectal microbiome of males and females with acute viremia and immune responses associated with protection were seen and point to effects of the microbiome on vaccine-induced immunity and viremia control. Our study clearly demonstrates direct effects of a mucosal SIV vaccine regimen on the rectal microbiome and validates our previously reported SIV vaccine-induced sex bias. Sex and the microbiome are critical factors that should not be overlooked in vaccine design and evaluation. IMPORTANCE Differences in HIV pathogenesis between males and females, including immunity postinfection, have been well documented, as have steroid hormone effects on the microbiome, which is known to influence mucosal immune responses. Few studies have applied this knowledge to vaccine trials. We investigated two SIV vaccine regimens combining mucosal priming immunizations and systemic protein boosting. We again report a vaccine-induced sex bias, with female rhesus macaques but not males displaying significantly reduced acute viremia. The vaccine regimens, especially the mucosal primes, significantly altered the rectal microbiome. The greatest effects were in females. Striking differences between female and male macaques in correlations of prevalent rectal bacteria with viral loads and potentially protective immune responses were observed. Effects of the microbiome on vaccine-induced immunity and viremia control require further study by microbiome transfer. However, the findings presented highlight the critical importance of considering effects of sex and the microbiome in vaccine design and evaluation.

Background IL-15 is a cytokine which stimulates the proliferation and cytotoxic function of CD8+ T and NK cells. We have produced and applied the native heterodimeric IL-15 (hetIL-15) on several preclinical models, which have supported the anti-tumor activity of hetIL-15. Based on these results, hetIL-15 has advanced to clinical trials. The objectives of this study were to explore how hetIL-15 shapes the tumor microenviroment and to characterize the interactions between tumor-infiltrating lymphoid and myeloid cells. Methods We studied the efficacy of locoregional administration of heterodimeric IL-15 (hetIL-15) in two different orthotopic triple-negative breast cancer (TNBC) mouse models, syngeneic for C57BL/6 and Balb/c, respectively. The effects of hetIL-15 on immune cells were analyzed by flow cytometry, immunohistochemistry (IHC) and gene expression profiling. The profile of the novel infiltrated dendritic cell populations was further explored by bulk and single cell RNAseq. Results hetIL-15 resulted in tumor eradication in 40% of treated mice and reduction of metastasis. Subsequent rechallenges with the same cell line failed to generate tumor regrowth, suggesting the development of immunological memory in hetIL-15 treated mice. hetIL-15 promoted tumor accumulation of proliferating and cytotoxic CD8+ T and NK cells. Additionally, peritumoral hetIL-15 administration resulted in an increased tumor infiltration of both conventional type 1 dendritic cells (cDC1s) and of a novel DC population found only in the hetIL-15 treated animals. Phenotypic profile analysis confirmed the expression of several cDC1 specific markers, including CD103 and IRF8 on this DC population.Transcriptomics and flow analysis of intratumoral dendritic cells indicate that the new hetIL-15 induced cells reside preferentially in the tumors and are distinct from cDC1 and cDC2 populations. Both cDC1s and the novel DC population were inversely correlated with the tumor size. Conclusions Locoregional administration of hetIL-15 results in complete eradication of EO771 and significant reduction of 4T1 primary breast cancer tumors, prolonged survival and long-lasting specific anti-tumor immunity. hetIL-15 increases the tumor infiltration of activated T and NK cells and intensifies the tumor infiltration of conventional type 1 dendritic cells (cDC1) and a new population of dendritic cells. We propose that the anti-cancer activity of hetIL-15 in primary EO771 tumors is orchestrated by the interplay of NK, CD8+T cells, cDC1 and a novel subset of DCs with a distinct phenotypic profile. These findings suggest a role for hetIL-15 in the treatment of breast cancer. Ethics Approval The study was approved by the National Cancer Institute-Frederick Animal Care and Use Committee, approval number 19–324 and was conducted in accordance with the ACUC guidelines and the NIH Guide for the Care and Use of Laboratory Animals.

Reliable detection and quantification of antigen-specific T cells are critical for assessing the immunogenicity of vaccine candidates. In this chapter, we describe the use of ELISpot and flow cytometry-based assays for efficient detection, mapping, and functional characterization of memory T lymphocytes in different tissues of rhesus macaques immunized with plasmid DNA. Flow cytometric assays provide a large amount of information, both phenotypic and functional, about individual cells, while the ELISpot is well suited for high throughput sample screening.

Reliable detection and quantification of antigen-specific T cells are critical for assessing the immunogenicity of vaccine candidates. In this chapter, we describe the use of ELISpot and flow cytometry-based assays for efficient detection, mapping, and functional characterization of memory T lymphocytes in different tissues of rhesus macaques immunized with plasmid DNA. Flow cytometric assays provide a large amount of information, both phenotypic and functional, about individual cells, while the ELISpot is well suited for high throughput sample screening.

Summary We compare immunogenicity and protective efficacy of an HIV vaccine comprised of env and gag DNA and Env (Envelope) proteins by co-administration of the vaccine components in the same muscles or by separate administration of DNA + protein in contralateral sites in female rhesus macaques. The 6-valent vaccine includes gp145 Env DNAs, representing six sequentially isolated Envs from the HIV-infected individual CH505, and matching GLA-SE-adjuvanted gp120 Env proteins. Interestingly, only macaques in the co-administration vaccine group are protected against SHIV CH505 acquisition after repeated low-dose intravaginal challenge and show 67% risk reduction per exposure. Macaques in the co-administration group develop higher Env-specific humoral and cellular immune responses. Non-neutralizing Env antibodies, ADCC, and antibodies binding to FcγRIIIa are associated with decreased transmission risk. These data suggest that simultaneous recognition, processing, and presentation of DNA + Env protein in the same draining lymph nodes play a critical role in the development of protective immunity.

Anti-retroviral therapy (ART) has been highly successful in controlling HIV replication, reducing viral burden, and preventing both progression to AIDS and viral transmission. Yet, ART alone cannot cure the infection. Even after years of successful therapy, ART withdrawal leads inevitably to viral rebound within a few weeks or months. Our hypothesis: effective therapy must control both the replicating virus pool and the reactivatable latent viral reservoir. To do this, we have combined ART and immunotherapy to attack both viral pools simultaneously. The vaccine regimen consisted of DNA vaccine expressing SIV Gag, followed by a boost with live attenuated rubella/gag vectors. The vectors grow well in rhesus macaques, and they are potent immunogens when used in a prime and boost strategy. We infected rhesus macaques by high dose mucosal challenge with virulent SIVmac251 and waited three days to allow viral dissemination and establishment of a reactivatable viral reservoir before starting ART. While on ART, the control group received control DNA and empty rubella vaccine, while the immunotherapy group received DNA/gag prime, followed by boosts with rubella vectors expressing SIV gag over 27 weeks. Both groups had a vaccine "take" to rubella, and the vaccine group developed antibodies and T cells specific for Gag. Five weeks after the last immunization, we stopped ART and monitored virus rebound. All four control animals eventually had a viral rebound, and two were euthanized for AIDS. One control macaque did not rebound until 2 years after ART release. In contrast, there was only one viral rebound in the vaccine group. Three out of four vaccinees had no viral rebound, even after CD8 depletion, and they remain in drug-free viral remission more than 2.5 years later. The strategy of early ART combined with immunotherapy can produce a sustained SIV remission in macaques and may be relevant for immunotherapy of HIV in humans.