DNA methyltransferase inhibition overcomes diphthamide pathway deficiencies underlying CD123-targeted treatment resistance.

The interleukin-3 receptor α subunit, CD123, is expressed in many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp's ability to ADP-ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies.

Antigen expression determines adenoviral vaccine potency independent of IFN and STING signaling.

Recombinant adenoviral vectors (rAds) are lead vaccine candidates for protection against a variety of pathogens, including Ebola, HIV, tuberculosis, and malaria, due to their ability to potently induce T cell immunity in humans. However, the ability to induce protective cellular immunity varies among rAds. Here, we assessed the mechanisms that control the potency of CD8 T cell responses in murine models following vaccination with human-, chimpanzee-, and simian-derived rAds encoding SIV-Gag antigen (Ag). After rAd vaccination, we quantified Ag expression and performed expression profiling of innate immune response genes in the draining lymph node. Human-derived rAd5 and chimpanzee-derived chAd3 were the most potent rAds and induced high and persistent Ag expression with low innate gene activation, while less potent rAds induced less Ag expression and robustly induced innate immunity genes that were primarily associated with IFN signaling. Abrogation of type I IFN or stimulator of IFN genes (STING) signaling increased Ag expression and accelerated CD8 T cell response kinetics but did not alter memory responses or protection. These findings reveal that the magnitude of rAd-induced memory CD8 T cell immune responses correlates with Ag expression but is independent of IFN and STING and provide criteria for optimizing protective CD8 T cell immunity with rAd vaccines.

The stem of vesicular stomatitis virus G can be replaced with the HIV-1 Env membrane-proximal external region without loss of G function or membrane-proximal external region antigenic properties.

The structure of the HIV-1 envelope membrane-proximal external region (MPER) is influenced by its association with the lipid bilayer on the surface of virus particles and infected cells. To develop a replicating vaccine vector displaying MPER sequences in association with membrane, Env epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, or both were grafted into the membrane-proximal stem region of the vesicular stomatitis virus (VSV) glycoprotein (G). VSV encoding functional G-MPER chimeras based on G from the Indiana or New Jersey serotype propagated efficiently, although grafting of both epitopes (G-2F5-4E10) modestly reduced replication and resulted in the acquisition of one to two adaptive mutations in the grafted MPER sequence. Monoclonal antibodies 2F5 and 4E10 efficiently neutralized VSV G-MPER vectors and bound to virus particles in solution, indicating that the epitopes were accessible in the preattachment form of the G-MPER chimeras. Overall, our results showed that the HIV Env MPER could functionally substitute for the VSV G-stem region implying that both perform similar functions even though they are from unrelated viruses. Furthermore, we found that the MPER sequence grafts induced low but detectable MPER-specific antibody responses in rabbits vaccinated with live VSV, although additional vector and immunogen modifications or use of a heterologous prime-boost vaccination regimen will be required to increase the magnitude of the immune response.

Coadministration of polyinosinic:polycytidylic acid and immunostimulatory complexes modifies antigen processing in dendritic cell subsets and enhances HIV gag-specific T cell immunity.

Currently approved adjuvants induce protective Ab responses but are more limited for generating cellular immunity. In this study, we assessed the effect of combining two adjuvants with distinct mechanisms of action on their ability to prime T cells: the TLR3 ligand, polyinosinic:polycytidylic acid (poly I:C), and immunostimulatory complexes (ISCOMs). Each adjuvant was administered alone or together with HIV Gag protein (Gag), and the magnitude, quality, and phenotype of Gag-specific T cell responses were assessed. For CD8 T cells, all adjuvants induced a comparable response magnitude, but combining poly I:C with ISCOMs induced a high frequency of CD127(+), IL-2-producing cells with decreased expression of Tbet compared with either adjuvant alone. For CD4 T cells, combining poly I:C and ISCOMs increased the frequency of multifunctional cells, producing IFN-γ, IL-2, and TNF, and the total magnitude of the response compared with either adjuvant alone. CD8 or CD4 T cell responses induced by both adjuvants mediated protection against Gag-expressing Listeria monocytogenes or vaccinia viral infections. Poly I:C and ISCOMs can alter Ag uptake and/or processing, and we therefore used fluorescently labeled HIV Gag and DQ-OVA to assess these mechanisms, respectively, in multiple dendritic cell subsets. Poly I:C promoted uptake and retention of Ag, whereas ISCOMs enhanced Ag degradation. Combining poly I:C and ISCOMs caused substantial death of dendritic cells but persistence of degraded Ag. These data illustrate how combining adjuvants, such as poly I:C and ISCOMs, that modulate Ag processing and have potent innate activity, can enhance the magnitude, quality, and phenotype of T cell immunity.

SIV antigen-specific effects on immune responses induced by vaccination with DNA electroporation and plasmid IL-12.

Molecular adjuvants are important for augmenting or modulating immune responses induced by DNA vaccination. Promising results have been obtained using IL-12 expression plasmids in a variety of disease models including the SIV model of HIV infection. We used a mouse model to evaluate plasmid IL-12 (pIL-12) in a DNA prime, recombinant adenovirus serotype 5 (rAd5) boost regimen specifically to evaluate the effect of IL-12 expression on cellular and humoral immunity induced against both SIVmac239 Gag and Env antigens. Priming with electroporated (EP) DNA+pIL-12 resulted in a 2-4-fold enhanced frequency of Gag-specific CD4 T cells which was maintained through the end of the study irrespective of the pIL-12 dose, while memory Env-specific CD4+T cells were maintained only at the low dose of pIL-12. There was little positive effect of pIL-12 on the humoral response to Env, and in fact, high dose pIL-12 dramatically reduced SIV Env-specific IgG. Additionally, both doses of pIL-12 diminished the frequency of CD8 T-cells after DNA prime, although a rAd5 boost recovered CD8 responses regardless of the pIL-12 dose. In this prime-boost regimen, we have shown that a high dose pIL-12 can systemically reduce Env-specific humoral responses and CD4T cell frequency, but not Gag-specific CD4+ T cells. These data indicate that it is important to independently characterize individual SIV or HIV antigen immunogenicity in multi-antigenic vaccines as a function of adjuvant dose.

Comparative analysis of the magnitude, quality, phenotype, and protective capacity of simian immunodeficiency virus gag-specific CD8+ T cells following human-, simian-, and chimpanzee-derived recombinant adenoviral vector immunization.

Recombinant adenoviral vectors (rAds) are the most potent recombinant vaccines for eliciting CD8(+) T cell-mediated immunity in humans; however, prior exposure from natural adenoviral infection can decrease such responses. In this study we show low seroreactivity in humans against simian- (sAd11, sAd16) or chimpanzee-derived (chAd3, chAd63) compared with human-derived (rAd5, rAd28, rAd35) vectors across multiple geographic regions. We then compared the magnitude, quality, phenotype, and protective capacity of CD8(+) T cell responses in mice vaccinated with rAds encoding SIV Gag. Using a dose range (1 × 10(7)-10(9) particle units), we defined a hierarchy among rAd vectors based on the magnitude and protective capacity of CD8(+) T cell responses, from most to least, as: rAd5 and chAd3, rAd28 and sAd11, chAd63, sAd16, and rAd35. Selection of rAd vector or dose could modulate the proportion and/or frequency of IFN-γ(+)TNF-α(+)IL-2(+) and KLRG1(+)CD127(-)CD8(+) T cells, but strikingly ∼30-80% of memory CD8(+) T cells coexpressed CD127 and KLRG1. To further optimize CD8(+) T cell responses, we assessed rAds as part of prime-boost regimens. Mice primed with rAds and boosted with NYVAC generated Gag-specific responses that approached ∼60% of total CD8(+) T cells at peak. Alternatively, priming with DNA or rAd28 and boosting with rAd5 or chAd3 induced robust and equivalent CD8(+) T cell responses compared with prime or boost alone. Collectively, these data provide the immunologic basis for using specific rAd vectors alone or as part of prime-boost regimens to induce CD8(+) T cells for rapid effector function or robust long-term memory, respectively.

Type I IFN induced by adenovirus serotypes 28 and 35 has multiple effects on T cell immunogenicity.

Recombinant adenovirus (rAd) vectors are being investigated as vaccine delivery vehicles in preclinical and clinical studies. rAds constructed from different serotypes differ in receptor usage, tropism, and ability to activate cells, aspects of which likely contribute to their different immunogenicity profiles. In this study, we compared the infectivity and cell stimulatory capacity of recombinant adenovirus serotype 5 (rAd5), recombinant adenovirus serotype 28 (rAd28), and recombinant adenovirus serotype 35 (rAd35) in association with their respective immunogenicity profiles. We found that rAd28 and rAd35 infected and led to the in vitro maturation and activation of both human and mouse dendritic cells more efficiently compared with rAd5. In stark contrast to rAd5, rAd28 and rAd35 induced production of IFN-α and stimulated IFN-related intracellular pathways. However, the in vivo immunogenicity of rAd28 and rAd35 was significantly lower than that of rAd5. Deletion of IFN-α signaling during vaccination with rAd28 and rAd35 vectors increased the magnitude of the insert-specific T cell response to levels induced by vaccination with rAd5 vector. The negative impact of IFN-α signaling on the magnitude of the T cell response could be overcome by increasing the vaccine dose, which was also associated with greater polyfunctionality and a more favorable long-term memory phenotype of the CD8 T cell response in the presence of IFN-α signaling. Taken together, our results demonstrate that rAd-induced IFN-α production has multiple effects on T cell immunogenicity, the understanding of which should be considered in the design of rAd vaccine vectors.

CD8+ T cell responses following replication-defective adenovirus serotype 5 immunization are dependent on CD11c+ dendritic cells but show redundancy in their requirement of TLR and nucleotide-binding oligomerization domain-like receptor signaling.

Replication-defective adenovirus serotype 5 (rAd5) is the most potent recombinant vector for eliciting CD8 T cell responses in humans. In this study, the innate mechanisms that influence T cell responses following rAd5 immunization were assessed in mice. Using rAd5 expressing enhanced GFP (eGFP-rAd5), we show that rAd5 transfects CD11c(+) dendritic cells (DCs) in draining lymph nodes in vivo following s.c. or i.m. immunization. Among distinct DC subsets, eGFP expression was highest in CD11c(+)CD8(-)B220(-) with a lower frequency detected in CD11c(+)CD8(+)B220(-) and CD11c(+)B220(+) plasmacytoid DCs. CD11c(+) DCs but not CD11c(-) cells from mice immunized with rAd5 encoding the SIINFEKL peptide induced proliferation of naive OT-I CD8 T cells. Furthermore, CD11c(+)CD8(+)B220(-) was the most potent DC subset for eliciting naive OT-I CD8 T cell proliferation. Of note, mice with pre-existing immunity to rAd5 had a substantial decrease in eGFP expression in DCs, which was associated with approximately 2-fold decrease in Th1 and complete inhibition of CD8 responses. Thus, pre-existing rAd5 immunity has a greater influence on CD8 compared with CD4 T cell responses. In terms of how innate cytokines and signaling pathways influenced T cell immunity following rAd5 immunization, we show that the magnitude and quality of CD8 T cell responses are partially dependent on MyD88 but independent of IL-12, type I IFN, apoptosis-associated speck-like protein, nucleotide-binding oligomerization domain-like receptor protein 3, and IL-1. Taken together, these data demonstrate a critical role for CD11c(+) DCs for CD8 responses but striking redundancy for innate cytokines and signaling by TLR and nucleotide-binding oligomerization domain-like receptor pathways.

IL-10 production differentially influences the magnitude, quality, and protective capacity of Th1 responses depending on the vaccine platform.

The quality of a Th1 response can be a prospective correlate of vaccine-mediated protection against certain intracellular pathogens. Using two distinct vaccine platforms, we evaluate the influence of interleukin (IL) 10 production on the magnitude, quality, and protective capacity of CD4(+) T cell responses in the mouse model of Leishmania major infection. Multiparameter flow cytometry was used to delineate the CD4(+) T cell production of interferon (IFN) gamma, IL-2, tumor necrosis factor (TNF), and IL-10 (or combinations thereof) after vaccination. Immunization with a high dose of adenovirus (ADV) expressing leishmanial proteins (MML-ADV) elicited a limited proportion of multifunctional IFN-gamma(+)IL-2(+)TNF(+) Th1 cells, a high frequency of IL-10-producing CD4(+) T cells, and did not protect against subsequent challenge. Surprisingly, in the absence of IL-10, there was no change in the magnitude, quality, or protective capacity of the Th1 response elicited by high-dose MML-ADV. In contrast, after immunization with MML protein and CpG (MML + CpG), IL-10 limited the production of IL-12 by DCs in vivo, thereby decreasing the generation of multifunctional Th1 cells. Consequently, three immunizations with MML + CpG were required for full protection. However, inhibiting IL-10 at the time of immunization enhanced the magnitude and quality of the Th1 response sufficiently to mediate protection after only a single immunization. Overall, we delineate distinct mechanisms by which vaccines elicit protective Th1 responses and underscore the importance of multifunctional CD4(+) T cells.

Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major.

CD4+ T cells have a crucial role in mediating protection against a variety of pathogens through production of specific cytokines. However, substantial heterogeneity in CD4+ T-cell cytokine responses has limited the ability to define an immune correlate of protection after vaccination. Here, using multiparameter flow cytometry to assess the immune responses after immunization, we show that the degree of protection against Leishmania major infection in mice is predicted by the frequency of CD4+ T cells simultaneously producing interferon-gamma, interleukin-2 and tumor necrosis factor. Notably, multifunctional effector cells generated by all vaccines tested are unique in their capacity to produce high amounts of interferon-gamma. These data show that the quality of a CD4+ T-cell cytokine response can be a crucial determinant in whether a vaccine is protective, and may provide a new and useful prospective immune correlate of protection for vaccines based on T-helper type 1 (TH1) cells.