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Cancer is becoming increasingly understood not only as a disease of pathological cells but also as one of immune hypofunction. The heterogenous and patient-specific nature of cancer further underscores the need for personalized cellular therapies, which are currently produced ex vivo. Gene-modulating approaches, such as therapeutic RNAs and improved viral vectors, now bring us closer toward strategies for mitigating disease, particularly for diseases that benefit from altering gene or transgene expression profiles in pathological or therapeutic immune cells. An advancing toolbox of technologies and trends toward simplifying personalized therapies foreshadow opportunities for direct, in vivo precision medicine against cancer. [ABSTRACT FROM AUTHOR]

The genetic diversity of HIV-1 represents a major challenge in vaccine development. In this study, we estab-lish a rationale for eliminating HIV-1-infected cells by targeting cellular immune responses against stable human endogenous retroviral (HERV) antigens. HERV DNA sequences in the human genome represent the remnants of ancient infectious retroviruses. We show that the infection of [CD4.sup.+] T cells with HIV-1 resulted in transcription of the HML-2 lineage of HERV type K [HERV-K(HML-2)] and the expression of Gag and Env proteins. HERV-K(HML-2)-specific CD8+ T cells obtained from HIV-1-infected human subjects responded to HIV-1-infected cells in a Vif-dependent manner in vitro. Consistent with the proposed mode of action, a HERV-K(HML-2)-specific [CD8.sup.+] T cell clone exhibited comprehensive elimination of cells infected with a panel of globally diverse HIV-1, HIV-2, and SIV isolates in vitro. We identified a second T cell response that exhibited cross-reactivity between homologous HIV-1-Pol and HERV-K(HML-2)-Pol determinants, raising the possibility that homology between HIV-1 and HERVs plays a role in shaping, and perhaps enhancing, the T cell response to HIV-1. This justifies the consideration of HERV-K(HML-2)-specific and cross-reactive T cell responses in the natural control of HIV-1 infection and for exploring HERV-K(HML-2)-targeted HIV-1 vaccines and immunotherapeutics., Introduction The genetic diversity of HIV-1 is considerable, with amino acids in Env differing by as much as 20% within a subtype and by more than 35% between subtypes and [...]

The enormous sequence diversity of HIV remains a major roadblock to the development of a prophylactic vaccine and new approaches to induce protective immunity are needed. Endogenous retrotransposable elements (ERE) such as endogenous retrovirus K (ERV)-K and long interspersed nuclear element-1 (LINE-1) are activated during HIV-1-infection and could represent stable, surrogate targets to eliminate HIV-1-infected cells. Here, we explored the hypothesis that vaccination against ERE would protect macaques from acquisition and replication of simian immunodeficiency virus (SIV). Following vaccination with antigens derived from LINE-1 and ERV-K consensus sequences, animals mounted immune responses that failed to delay acquisition of SIVsmE660. We observed no differences in acute or set point viral loads between ERE-vaccinated and control animals suggesting that ERE-specific responses were not protective. Indeed, ERE-specific T cells failed to expand anamnestically in vivo following infection with SIVsmE660 and did not recognize SIV-infected targets in vitro, in agreement with no significant induction of targeted ERE mRNA by SIV in macaque CD4+ T cells. Instead, lower infection rates and viral loads correlated significantly to protective TRIM5α alleles. Cumulatively, these data demonstrate that vaccination against the selected ERE consensus sequences in macaques did not lead to immune-mediated recognition and killing of SIV-infected cells, as has been shown for HIV-infected human cells using patient-derived HERV-K-specific T cells. Thus, further research is required to identify the specific nonhuman primate EREs and retroviruses that recapitulate the activity of HIV-1 in human cells. These results also highlight the complexity in translating observations of the interplay between HIV-1 and human EREs to animal models. [ABSTRACT FROM AUTHOR]

Protection against mucosally transmitted infections probably requires immunity at the site of pathogen entry, yet there are no mucosal adjuvant formulations licensed for human use. Polyethyleneimine (PEI) represents a family of organic polycations used as nucleic acid transfection reagents in vitro and DNA vaccine delivery vehicles in vivo. Here we show that diverse PEI forms have potent mucosal adjuvant activity for viral subunit glycoprotein antigens. A single intranasal administration of influenza hemagglutinin or herpes simplex virus type-2 (HSV-2) glycoprotein D with PEI elicited robust antibody-mediated protection from an otherwise lethal infection, and was superior to existing experimental mucosal adjuvants. PEI formed nanoscale complexes with antigen, which were taken up by antigen-presenting cells in vitro and in vivo, promoted dendritic cell trafficking to draining lymph nodes and induced non-proinflammatory cytokine responses. PEI adjuvanticity required release of host double-stranded DNA that triggered Irf3-dependent signaling. PEI therefore merits further investigation as a mucosal adjuvant for human use. [ABSTRACT FROM AUTHOR]

Abstract: Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly related Env surface (gp120) antigens produced in different systems: (a) mammalian (293 FreeStyle™ cells; 293F) cells in the presence of kifunensine, which impart only high-mannose glycans; (b) insect cells (Spodoptera frugiperda, Sf9), which confer mainly paucimannosidic glycans; (c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic™), which impart high-mannose, hybrid and complex glycans without sialic acid; and (d) 293F cells, which impart high-mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9-derived and wild-type mammalian-cell-derived material that is predicted to affect ligand binding sites proximal to glycans. Modeling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4 binding and CD4-induced sites compared to those expressed in the system that do, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic-acid-containing gp120 was significantly more immunogenic than the sialylated version when administered in two different adjuvants, and induced higher titers of antibodies competing for CD4 binding site ligand–gp120 interaction. These findings suggest that non-sialic-acid-imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations that should be considered when selecting expression systems for glycosylated antigens to be used for structure–function studies and for vaccine use. [Copyright &y& Elsevier]

It has recently been shown that polymorphism at the rhesus macaque TRIM5 locus can affect simian immunodeficiency virus (SIV) replication. Here we show that TRIM5 alleles can also affect acquisition of SIVsmE660. Animals coexpressing the TRIM5TFP and TRIM5CypA alleles took significantly longer to become infected with SIVsmE660, but not SIVmac239, after repeated limiting-dose intrarectal challenge than did animals expressing other TRIM5 allele combinations. Our results indicate that the TRIM5 alleles can be a barrier to productive infection and that this should be taken into account when designing acquisition studies using SIVsmE660 or related viruses. [ABSTRACT FROM AUTHOR]

Multiple myeloma is a largely incurable and life-threatening malignancy of antibody-secreting plasma cells. An effective and widely available animal model that recapitulates human myeloma and related plasma cell disorders is lacking. We show that busulfan-conditioned human IL-6-transgenic (hIL-6-transgenic) NSG (NSG+hIL6) mice reliably support the engraftment of malignant and premalignant human plasma cells, including from patients diagnosed with monoclonal gammopathy of undetermined significance, pre- and postrelapse myeloma, plasma cell leukemia, and amyloid light chain amyloidosis. Consistent with human disease, NSG+hIL6 mice engrafted with patient-derived myeloma cells developed serum M spikes, and a majority developed anemia, hypercalcemia, and/or bone lesions. Single-cell RNA sequencing showed nonmalignant and malignant cell engraftment, the latter expressing a wide array of mRNAs associated with myeloma cell survival and proliferation. Myeloma-engrafted mice given CAR T cells targeting plasma cells or bortezomib experienced reduced tumor burden. Our results establish NSG+hIL6 mice as an effective patient-derived xenograft model for study and preclinical drug development of multiple myeloma and related plasma cell disorders.

Natural killer (NK) cells are frequently expanded for the clinic using irradiated, engineered K562 feeder cells expressing a core transgene set of membrane-bound (mb) IL15 and/or mbIL21 together with 41BBL. Prior comparisons of mbIL15 to mbIL21 for NK expansion lack comparisons of key attributes of the resulting NK cells, including their high-dimensional phenotype, polyfunctionality, the breadth and potency of cytotoxicity, cellular metabolism, and activity in xenograft tumor models. Moreover, despite multiple rounds of K562 stimulation, studies of sequential use of mbIL15- and mbIL21-based feeder cells are absent. We addressed these gaps and found that using mbIL15- versus mbIL21-based feeder cells drove distinct phenotypic and functional profiles. Feeder cells expressing mbIL15 alone drove superior functionality by nearly all measures, whereas those expressing mbIL21 alone drove superior yield. In combination, most attributes resembled those imparted by mbIL21, whereas in sequence, NK yield approximated that imparted by the first cytokine, and the phenotype, transcriptome, and function resembled that driven by the second cytokine, highlighting the plasticity of NK cell differentiation. The sequence mbIL21 followed by mbIL15 was advantageous in achieving significant yields of highly functional NK cells that demonstrated equivalent in vivo activity to those expanded by mbIL15 alone in two of three xenograft models. Our findings define the impact of mbIL15 versus mbIL21 during NK expansion and reveal a previously underappreciated tradeoff between NK yield and function for which sequential use of mbIL21-based followed by mbIL15-based feeder cells may be the optimal approach in many settings., (©2023 The Authors; Published by the American Association for Cancer Research.)

Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable success in the treatment of hematologic malignancies. Unfortunately, it has limited efficacy against solid tumors, even when the targeted antigens are well expressed. A better understanding of the underlying mechanisms of CAR T-cell therapy resistance in solid tumors is necessary to develop strategies to improve efficacy. Here we report that solid tumors release small extracellular vesicles (sEV) that carry both targeted tumor antigens and the immune checkpoint protein PD-L1. These sEVs acted as cell-free functional units to preferentially interact with cognate CAR T cells and efficiently inhibited their proliferation, migration, and function. In syngeneic mouse tumor models, blocking tumor sEV secretion not only boosted the infiltration and antitumor activity of CAR T cells but also improved endogenous antitumor immunity. These results suggest that solid tumors use sEVs as an active defense mechanism to resist CAR T cells and implicate tumor sEVs as a potential therapeutic target to optimize CAR T-cell therapy against solid tumors., Significance: Small extracellular vesicles secreted by solid tumors inhibit CAR T cells, which provide a molecular explanation for CAR T-cell resistance and suggests that strategies targeting exosome secretion may enhance CAR T-cell efficacy. See related commentary by Ortiz-Espinosa and Srivastava, p. 2637., (©2023 American Association for Cancer Research.)

A fundamental limitation of T cell therapies in solid tumors is loss of inflammatory effector functions, such as cytokine production and proliferation. Here, we target a regulatory axis of T cell inflammatory responses, Regnase-1 and Roquin-1, to enhance antitumor responses in human T cells engineered with two clinical-stage immune receptors. Building on previous observations of Regnase-1 or Roquin-1 knockout in murine T cells or in human T cells for hematological malignancy models, we found that knockout of either Regnase-1 or Roquin-1 alone enhances antitumor function in solid tumor models, but that knockout of both Regnase-1 and Roquin-1 increases function further than knockout of either regulator alone. Double knockout of Regnase-1 and Roquin-1 increased resting T cell inflammatory activity and led to at least an order of magnitude greater T cell expansion and accumulation in xenograft mouse models, increased cytokine activity, and persistence. However double knockout of Regnase-1 and Roguin-1 also led to a lymphoproliferative syndrome and toxicity in some mice. These results suggest that regulators of immune inflammatory functions may be interesting targets to modulate to improve antitumor responses.

Viral engineered chimeric antigen receptor (CAR) T cell therapies are potent, targeted cancer immunotherapies, but their permanent CAR expression can lead to severe adverse effects. Nonviral messenger RNA (mRNA) CAR T cells are being explored to overcome these drawbacks, but electroporation, the most common T cell transfection method, is limited by cytotoxicity. As a potentially safer nonviral delivery strategy, here, sequential libraries of ionizable lipid nanoparticle (LNP) formulations with varied excipient compositions were screened in comparison to a standard formulation for improved mRNA delivery to T cells with low cytotoxicity, revealing B10 as the top formulation with a 3-fold increase in mRNA delivery. When compared to electroporation in primary human T cells, B10 LNPs induced comparable CAR expression with reduced cytotoxicity while demonstrating potent cancer cell killing. These results demonstrate the impact of excipient optimization on LNP performance and support B10 LNPs as a potent mRNA delivery platform for T cell engineering.

The expression of endogenous retrotransposable elements, including long interspersed nuclear element 1 (LINE-1 or L1) and human endogenous retrovirus, accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. In this article, we address the safety and immunogenicity of vaccination with these elements. We used immunohistochemical analysis and literature precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 open reading frame 2 induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human LINE-1 open reading frame 2 (96% identity with macaque), as well as simian endogenous retrovirus-K Gag and Env, induced polyfunctional T cell responses to all Ags, and Ab responses to simian endogenous retrovirus-K Env. There were no adverse safety or pathological findings related to vaccination. These studies provide the first evidence, to our knowledge, that immune responses can be induced safely against this class of self-antigens and pave the way for investigation of them as HIV- or tumor-associated targets.

T-cell responses to human endogenous retrovirus (HERV) K(HML-2) Gag and Env were mapped in HIV-1-infected subjects using 15 mer peptides. Small peptide pools and high concentrations were used to maximize sensitivity. In the 23 subjects studied, only three bona fide HERV-K(HML-2)-specific responses were detected. At these high peptide concentrations, we detected false-positive responses, three of which were mapped to an HIV-1 Gag peptide contaminant. Thus, HERV-K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected by 15 mer peptide mapping.

Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.