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Heterologous arenavirus vector prime-boost overrules self-tolerance for efficient tumor-specific CD8 T cell attack

Summary Therapeutic vaccination regimens inducing clinically effective tumor-specific CD8+ T lymphocyte (CTL) responses are an unmet medical need. We engineer two distantly related arenaviruses, Pichinde virus and lymphocytic choriomeningitis virus, for therapeutic cancer vaccination. In mice, life-replicating vector formats of these two viruses delivering a self-antigen in a heterologous prime-boost regimen induce tumor-specific CTL responses up to 50% of the circulating CD8 T cell pool. This CTL attack eliminates established solid tumors in a significant proportion of animals, accompanied by protection against tumor rechallenge. The magnitude of CTL responses is alarmin driven and requires combining two genealogically distantly related arenaviruses. Vector-neutralizing antibodies do not inhibit booster immunizations by the same vector or by closely related vectors. Rather, CTL immunodominance hierarchies favor vector backbone-targeted responses at the expense of self-reactive CTLs. These findings establish an arenavirus-based immunotherapy regimen that allows reshuffling of immunodominance hierarchies and breaking self-directed tolerance for efficient tumor control.


Correspondence daniel.pinschewer@unibas.ch
In brief Therapeutic tumor vaccination should break self-tolerance. Assessing combinations of engineered arenavirus vectors, Bonilla et al. find that distantly related vector combinations reshuffle T cell immunodominance hierarchies to break self-tolerance and eliminate established solid tumors, whereas closely related vectors interfere because of immunodominance of anti-vector CD8 T cells rather than antibodies.

INTRODUCTION
Cytotoxic CD8 + T lymphocytes (CTLs) are central mediators of adaptive immunity. Tumor-infiltrating CTLs in several tumor types are associated with clinical outcome, 1-3 and pre-existing CTL infiltration may predict responsiveness to immune checkpoint inhibition. 4 Analogously, CTLs are key players in HIV elite control and hepatitis B virus clearance. [5][6][7] Therapeutic vaccination for CTL induction holds great promise for cancer therapy 8,9 but has delivered inconsistent therapeutic benefits, including failure of large clinical trials. [10][11][12][13] Despite induction of sizeable tumor antigen-specific CD8 + T cell frequencies by modalities such as adjuvanted peptides, 14 inefficient tumor infiltration has curtailed the clinical efficacy of these cells. 15,16 Delivering tumor-associated antigens (TAAs) in the context of virus-induced inflammation 17 has significant potential to overcome these hurdles. Accordingly, several viral vector platforms have been developed for therapeutic use against solid tumors. 13,[18][19][20][21][22][23][24][25] The immunostimulatory properties of the viral particles themselves, exhibiting pathogen-associated molecular patterns, activate antigen-presenting cells (APCs) to augment and differentiate immune responses. 26,27 In addition, certain replicating viral delivery systems trigger release of damage-associated molecular patterns or alarmins, such as interleukin-33 (IL-33). 28,29 These signals critically augment activated T cell expansion, effector differentiation, and anti-tumor efficacy. 30,31 Anti-vector immunity can inhibit viral delivery systems, impeding their re-administration to augment immune responses. A single immunization with vaccinia virus or modified vaccinia virus Ankara (MVA) elicits neutralizing antibody (nAb) responses in the majority of individuals 32 and inhibits vaccinia-vectored immunization. 33 The seroprevalence of common adenovirus (Ad) serotypes such as Ad5 can regionally exceed 90%. Ad5-nAbs dampen or even abrogate responses to recombinant Ad5-based vaccines, 34 and use of simian Ad (sAd) backbones with lower seroprevalence has increased immunological response rates in clinical trials. 35,36 Yet, also sAd vectors induce vector-specific nAbs when used for vaccination and have failed to demonstrate efficient homologous boosting capacity. 37 More surprisingly, even serologically distinct adenovectors did not efficiently boost each other 36,37 , and adenoviral vectors are now commonly combined with poxvirus-based platforms for heterologous primeboost vaccination. 37 For several decades, members of the Arenaviridae family have found widespread use in basic immunological research because of their capacity to induce CTL responses of exceptional magnitude, functionality, and longevity. 38 Reverse genetic techniques have enabled the tailored design of this virus family and its exploitation for vaccination. 39, 40 Replication-deficient vectors (rLCMV) based on the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) have demonstrated excellent CTL and nAb induction against vectorized transgenes in mice, non-human primates, and, recently, also in humans. [41][42][43][44] As an important differentiation from other viral vector technologies, rLCMVbased immunization only rarely induces vector-nAbs, facilitating the vector's repeated administration in homologous prime-boost vaccination. 41,43,44 This peculiarity of LCMV-based vectors is due to an N-linked ''glycan shield'' on the outer globular domain of the viral envelope glycoprotein domain that impairs antibody accessibility to critical neutralizing epitopes. 45 rLCMV vectors are currently in clinical phase 2 testing for prevention of cytomegalovirus-associated disease in transplant recipients. 46 We and others have developed replication-attenuated, trisegmented, arenavirus-based vector formats (artARENA, r3ARENA; Figures 1B and 1C) that induce even more potent effector CTL responses than replication-deficient rLCMV or commonly used poxvirus and Ad vector systems. 29,47,48 An LCMV-based artARENA vector (artLCMV) has recently entered early-stage clinical testing. 49 When used to deliver tumor selfantigens to tumor-bearing mice, artLCMV induced potent antitumor CTL responses and extended the animals' survival. 29 Tumor-specific CTL responses were, however, lower in magnitude than those induced against vectorized non-self-antigens, and complete tumor remission was not achieved. This suggested that self-tolerance limited the therapeutic efficacy of artLCMVbased immunization. 29 Here we systematically explore a range of additional arenavirus vector backbones for vaccine delivery. We report that, contrary to commonly held concepts, anti-vector CTL responses rather than nAbs curtail the immunogenicity of homologous arenavirus vector prime-boost vaccination. Accordingly, backbones of distant genealogic relationship offered the most potent heterologous prime-boost combinations, resulting in considerable rates of complete tumor rejection.

RESULTS
Arenavirus vector backbone candidates and their genetic and phenotypic stability Arenaviruses form enveloped particles and contain two segments of negative-stranded RNA. The large (L) segment encodes for the viral polymerase L and the matrix protein Z, whereas the short (S) segment carries the envelope glycoprotein (GP) and nucleoprotein (NP) genes, separated by intergenic regions, respectively ( Figure 1A). We and others have incorporated transgenic sequences such as TAAs into replicating arenavirus vectors by segregating the NP and GP genes onto artificially duplicated S segments (S NP and S GP ; Figures 1B and S1C). 29,47,48 This can be achieved by the artARENA (e.g., artLCMV) or r3ARENA (e.g., r3LCMV) design strategy ( Figures 1B and 1C). Here we vectorized additional mammalian arenaviruses (mammarenaviruses) to exploit their immunotherapeutic potential when combined in heterologous prime-boost combinations. Based on phylogenetic relationship, mammarenaviruses can be subdivided into the Old World group of viruses and four clades (A-D) of New World viruses ( Figure 1D). For vector generation (STAR methods), we selected three mutually very distantly related viruses representing the main branches of the phylogenetic tree: the prototypic Old World virus LCMV, the widely studied New World clade A virus Pichinde virus (PICV), and the Junin virus vaccine strain Candid#1 (CAND). CAND is in clinical use as a prophylactic vaccine against Junin virus, the causative agent of Argentine hemorrhagic fever, 50 and PICV has no human disease correlate but can infect humans, as documented in accidentally exposed laboratory workers. 51 LCMV infection is mostly asymptomatic or manifests as a flu-like infection. 52 Rare cases of choriomeningitis are generally self-limiting and, despite documented cases of protracted central nervous system manifestations, commonly heal without persisting sequelae. 53,54 From a safety show the mean ± SD of three cell culture wells (error bars mostly within symbol size). **p < 0.01 by unpaired two-tailed Student's t test.
(H-J) AGRAG mice were infected intravenously (i.v.) with the indicated viruses, and viremia was monitored by immunofocus assays detecting PICV-NP (H) or GFP (I) to calculate the NP:GFP infectivity ratio (J). Symbols represent means ± SD of 3-5 mice. (K-M) RT-PCR strategy (K) to amplify recombined WT-like PICV S segment RNA species re-uniting NP and GP sequences. Also shown is a gel electrophoresis image of RT-PCR products (L) obtained from serum samples collected at the indicated time points. Each lane represents an individual mouse. Sera from uninfected mice (''none'') and water were included as negative controls. Sequence analysis of the bands numbered in (L) suggested recombination products, as depicted schematically in (M). IGR, S segment intergenic region; UTR, untranslated region. perspective, all three viruses are valid vector backbone candidates for immunotherapy use in humans. We generated artAR-ENA (artLCMV, artPICV, and artCAND) as well as r3ARENA (r3LCMV, r3PICV, and r3CAND) vectors expressing fluorescent reporter proteins. In cell culture, artARENA vectors as well as their respective r3ARENA counterparts reached lower titers than their respective parental wild-type (WT) viruses ( Figures  1E-1G). These observations extended and generalized earlier findings of attenuated artARENA and r3ARENA vector growth. 29,47,48 Genetic and phenotypic stability are key criteria for the clinical utility of replicating viral vector systems. We have reported previously that artLCMV stably retained its genome organization and transgene expression over extended periods of in vivo replication, whereas r3LCMV underwent inter-segmental recombination, reverting to a non-transgenic bisegmented WT-like virus. 29 Here we used artPICV-GFP, r3PICV-GFP, and WT PICV (PICV-WT) to infect highly immunodeficient mice, which lack type I and type II interferon receptors and are devoid of T and B cells because of RAG deficiency (AGRAG mice). In the first 30 days of persistent infection, total viral loads (determined as PICV NP-expressing infectivity) in the blood of art-PICV-GFP-and r3PICV-GFP-infected animals were similar and below those of PICV-WT-infected controls ( Figure 1H). By day 70, however, r3PICV-GFP viremia exceeded the levels in artPICV-GFP-infected animals and increased continuously thereafter, eventually reaching levels equivalent to PICV-WT. Conversely, artPICV viremia remained consistently below PICV-WT controls. GFP transgene-expressing viral infectivity in r3PICV-GFP-and artPICV-GFP-infected mice was at comparable levels up to around day 70 of infection ( Figure 1I). Thereafter it declined continuously in r3PICV-GFP-infected animals but remained stable in artPICV-infected animals. The resulting ratio of total PICV infectivity to GFP-transgenic infectivity documented that r3PICV-GFP progressively lost its transgene, whereas transgene expression by artPICV-GFP remained stable throughout the observation period of more than 200 days ( Figure 1J). We performed RT-PCR to detect supposedly recombined WT-like S segments containing PICV NP and GP sequences ( Figure 1K). Such RNA species were absent from artPICV-GFP-infected mice but detected consistently in the blood of r3PICV-infected animals ( Figure 1L). Sequence analysis of amplicons revealed that some of them contained one or two GFP remnants flanked by partially or completely duplicated viral intergenic regions, identifying them as intersegmental recombination products of the S NP and S GP segments of r3PICV-GFP ( Figure 1M). On day 224 after infection, RT-PCR assays detected the S NP and S GP segments of artPICV-GFP but not the corresponding GFP-containing segments of r3PICV-GFP (Figures S1A-S1D), further supporting the notion of r3PICV-GFP transgene loss. When re-isolated after more than 200 days of persistent infection and propagated in cell culture, r3PICV-GFP reached titers equivalent to PICV-WT, whereas re-isolated artPICV-GFP growth was attenuated. This contrasted with the cell culture behavior of the inoculum of r3PICV-GFP and artPICV-GFP, both of which grew to lower titers than the corresponding PICV-WT ( Figure 1N). These findings indicated that the genetic instability of r3PICV-GFP was accompanied by phenotypic reversion to PICV-WT-like growth, whereas artPICV-GFP was genetically and phenotypically stable. Analogous findings were made with CAND-based vectors. S NP -S GP recombination products and loss of GFP-containing segments was also observed in r3CAND-infected AGRAG mice but not in artCAND-infected animals (Figures S1E-S1K). Re-isolated r3CAND-GFP exhibited CAND-WT-like cell culture growth behavior ( Figure S1L), whereas artCAND replication in AGRAG mice was at levels too low to allow virus re-isolation. These studies generalized the finding 29 that artARENA vectors are genetically and phenotypically stable, whereas r3ARENA vectors are prone to transgene loss and phenotypic reversion to a WT-like virus.
artARENA vectors are attenuated in guinea pig and mouse pathogenesis models Next we tested whether artPICV and artLCMV were attenuated in animal models. Guinea pigs were infected with titrated doses of PICV-WT, known to cause lethal disease in these animals, or with artPICV-E7E6 expressing a non-oncogenic fusion construct consisting of the HPV16 E7 and E6 open reading frames (ORFs) 55 (see chart in Figure 2A). Animals receiving diluent were included as a further control. At PICV-WT doses of 3 3 10e2 or 3 plaque-forming units (PFUs), three of eight animals reached humane endpoints (Figure 2A), a disease that was always accompanied by high-level viremia (>10e3 PFUs; Figures 2B and 2C). At the highest PICV-WT dose (3 3 10e4 PFUs), seven of eight guinea pigs developed high-level viremia and terminal disease (Figures 2A and 2D). In contrast, artPICV-E7E6 infection at 3 3 10e2 PFUs was aviremic ( Figure 2E), and doses of 3 3 10e4 or even 3 3 10e6 PFUs (100-fold higher than the highest PICV-WT dose tested) did not result in high-level viremia ( Figures 2F and 2G). Transient low-level viremia was detected in only 1 of 8 animals in both of these latter cohorts ( Figures 2F and 2G), and none of these animals developed terminal disease ( Figure 2A). When administered 3 3 10e2 PFUs of artPICV-E7E6, the lowest dose tested, seven of eight animals were free of disease throughout the 27-day observation period, and none of the animals had detectable viremia (Figure 2E). On day 24 after vector inoculation, one of these aviremic animals suddenly exhibited signs of disease corresponding to humane study endpoints, necessitating its euthanasia on day 25. The absence of detectable viral loads in this animal suggested, however, that its disease was unrelated to artPICV-E7E6 vector administration. Measurements of body weight loss, a commonly used parameter of PICV-induced disease in guinea pigs, provided additional independent support for the conclusion that artPICV-E7E6 was substantially attenuated ( Figure S2).
Intracranial inoculation of mice is the standard model to assess the neurovirulence of LCMV-based vectors. 29,41,56 In agreement with earlier data demonstrating attenuation of artLCMV vectors, 29 we found that 100-1,000 PFUs of artLCMV-E7E6 had to be administered intracranially to elicit terminal choriomeningitis in about half of the animals, whereas 1 PFU of LCMV-WT resulted in terminal disease in all mice (Figure 2I). LCMV-induced choriomeningitis is mediated by CD8 T cells, which attack virus-infected meningeal cells and astrocytes, resulting in blood-brain barrier (BBB) breakdown and brain edema. [57][58][59] To investigate the mechanisms underlying reduced artLCMV neurovirulence, we infected WT mice intracranially with 10 PFUs of artLCMV-E7E6 or LCMV-WT or with diluent. Seven days later, at the peak of disease in LCMV-WT-infected animals, we determined immunoglobulin deposits in the brain parenchyma as a surrogate of BBB integrity ( Figures 2J  and 2K). The brain area affected by BBB breakdown was significantly larger in LCMV-WT-than in artLCMV-E7E6-infected mice ( Figure 2L). Regions of dense immunoglobulin deposits were evident in LCMV-WT-infected brains, notably around the longitudinal fissure, with substantial extension into deeper cortical layers, whereas in artLCMV-E7E6-infected brains, only small immunoglobulin deposits were detected in proximity to the longitudinal fissure. This morphological correlate of reduced immunopathological damage furthers our understanding of attenuated artLCMV neurovirulence. 57 Immunogenicity and epitope dominance in heterologous artARENA prime-boost vaccination Next we tested the utility of artPICV-artLCMV as a heterologous prime-boost regimen. We primed C57BL/6 mice with artLCMV-E7E6 or artPICV-E7E6, followed by artLCMV-E7E6 boost on day 13. E7-specific CTL responses in the blood on day 9 were somewhat higher upon artLCMV-E7E6 prime than after art-PICV-E7E6 prime ( Figure 3A). Seven days after heterologous artLCMV-E7E6 boost (day 20), artPICV-E7E6-primed animals reached E7-specific CTL frequencies of more than 50% of the total circulating CD8 + T cell pool, with only limited contraction over a 1-month-period ( Figures 3A-3C and S3). These CTL frequencies vastly exceeded those induced by homologous artLCMV-E7E6 prime-boost, which remained in the 6%-7% range, similar to the frequencies after prime. All of these responses were polyfunctional, as determined by interferon g (IFN-g), tumor necrosis factor alpha (TNF-a), and IL-2 secretion  Article ll OPEN ACCESS upon peptide stimulation and comprised E7-as well as E6-specific CTLs ( Figure S3). The majority of tumors, however, do not exhibit viral determinants such HPV E7 and E6, and therefore active immunization has to rely on other classes of TAAs for which immune responses can be affected by self-tolerance. Studies of the P815 mouse tumor cell-derived cancer-testis antigen P1A, for example, have shown that post-natal expression is restricted to spermatogonia, placenta, and thymic medullary epithelial cells, with the latter being key for central tolerance induction. 60,61 Accordingly, P1A knockout mice spontaneously rejected P815 tumors, mounting P1A-specific CD8 T cell responses of significantly higher magnitude and functional avidity than WT animals. 62 To test whether heterologous artARENA immunization facilitates breaking self-tolerance, we immunized BALB/c mice with P1A-expressing artLCMV-P1A or artPICV-P1A. By day 38 after prime, the P1A-specific responses induced by either vector had leveled off in the range of 2%-3% of circulating CD8 + T cells ( Figure 3F). Upon heterologous artLCMV-P1A boost, artPICV-P1A-primed mice mounted P1A-specific CTL responses exceeding 50% of the circulating CD8 + T cell pool, substantially higher than upon artLCMV-P1A homologous boost ( Figures 3F-3H). These heterologous prime-boost-induced CTL responses contracted slowly, with frequencies of more than 20% persisting in peripheral blood for more than 3 months ( Figure 3F). Besides vector transgene-specific responses (E7E6 and P1A), we also determined dominant vector backbone-specific responses directed against the NP-derived epitopes NP396 in C57BL/6 mice (H-2D b restricted) and NP118 in BALB/c mice (H-2L d restricted). Interestingly, the responses of C57BL/6 and BALB/c mice, respectively, were substantially lower in animals receiving heterologous artPICV-artLCMV immunization compared with homologous artLCMV prime-boost (Figures 3D and 3I). Based on these measurements we calculated the transgene:backbone epitope dominance ratio (E7:NP396 [ Figure 3E]; P1A:NP118 [ Figure 3J]). It was substantially higher in heterologous compared with homologous prime-boost immunization ($50-fold in Figure 3E; >200-fold in Figure 3J), indicating that heterologous prime-boost immunization biased vaccineinduced CTL responses toward transgene-derived epitopes. In the context of active immunization for immunotherapy, it is important to induce high-frequency CTL responses within a short time window. artPICV-P1A-artLCMV-P1A heterologous prime-boost, administered at an interval of only 4-10 days, induced P1A-specific CTL responses of higher frequencies than obtained with homologous prime-boost immunization given at the same interval ( Figures S4A-S4D). A trend of higher P1Aspecific CTL frequencies at longer prime-boost intervals (7-10 days compared with 4 days) was accompanied by an inverse trend in NP118 backbone-targeting responses. artPICV-P1A prime followed by artLCMV-P1A boost clearly outperformed the inverse sequence of administration when administered at in-tervals of 4-10 days (Figures S4A-S4D). The prime-boost regimen of artPICV followed by artLCMV rather than the inverse sequence of administration was therefore used for subsequent studies. Only when the interval between the two vaccinations was substantially longer was the artPICV-P1A boost of artLCMV-P1A-primed responses effective (Figures S4E and  S4F).
Phenotype of artARENA-induced CTLs and their dependence on IL-33-ST2 alarmin signaling Next, we determined how heterologous artPICV-artLCMV prime-boost influenced the magnitude and phenotype of P1Aspecific CTL populations. Seven days after boost, CD62L lo effector/effector memory cells dominated the responses of BALB/c mice to homologous artLCMV-P1A prime-boost and heterologous artPICV-P1A-artLCMV-P1A immunization ( Figures  4A-4D). Importantly, heterologous prime-boost elicited not only higher P1A-specific CD62L lo effector/effector memory CTL populations than homologous prime-boost but the CD62L hi central memory population was also more abundant ( Figures 4C and  4D). We studied the phenotype of both CTL subsets by determining the cells' expression of the surface markers KLRG1, CX3CR1, CD27, CD43, and CD127 and of the transcription factors Tcf-1, Tbet, and Eomes ( Figures 4E and S5G). Within the CD62L hi and CD62L lo subsets of CTLs, heterologous primeboost immunization promoted expression of the effector differentiation markers KLRG1, CX3CR1, and CD43, 63-67 with a concomitant reduction in the proportion of cells expressing the memory markers CD27 and CD127. 66,68 We also observed that CTLs emerging from heterologous prime-boost expressed higher average Tbet levels and lower levels of Tcf-1, further supporting the conclusion that heterologous prime-boost augmented the effector differentiation of CTLs. Irrespective of this relative effector differentiation bias, heterologous primeboost augmented not only the total number of CTLs expressing the effector differentiation markers KLRG1, CX3CR1, and CD43 but also the population of cells expressing memory precursor markers (CD27 and CD127) and the stemness-defining transcription factor Tcf-1 ( Figure 4F). 69 When analyzed 4 weeks after boost ( Figures S5A-S5F), most of the differences between CTLs emerging from homologous and heterologous prime-boost persisted but, overall, were less pronounced than on day 7 after boost. Prominent populations of effector-like memory populations, characterized by the marker combinations CD43 -CD27 -, CX3CR1 + CD27 -, and KLRG1 + CD27 -, 63-67 were particularly abundant upon heterologous prime-boost immunization (Figure S5G). These observations indicated that CTL responses induced by heterologous prime-boost differed from those emerging from homologous prime-boost primarily by their higher cellularity paired with more pronounced effector differentiation. SD) or the mean fluorescence intensity (MFI ± SD). Means were calculated from six mice per immunization group (A-F) or from three unimmunized controls (A). N = 2. **p < 0.01 by unpaired two-tailed Student's t test.
IL-33 signals through its receptor ST2 are key for protective CTL responses to replicating viruses, 31 and IL-33 signals critically augment CTL responses to artLCMV-vectored immunization and the resulting tumor control. 29 Here, we compared the ST2 dependence of artARENA-induced CTL responses by immunizing ST2-deficient and WT control mice with artLCMV-E7E6, artPICV-E7E6, or artCAND-E7E6. artLCMV-and art-PICV-induced CTL responses to E7 were significantly lower in the blood of ST2-deficient mice than in WT controls ( Figures  4G and 4H). Conversely, ST2 deficiency had no clear effect on artCAND-induced CTL responses. ST2-dependent differences in artLCMV-and artPICV-induced CTL responses were also evident when enumerating IFN-g and TNF-a co-producing CTLs in the spleen ( Figure S6). These findings indicated that, besides artLCMV, artPICV-but not artCAND-induced CTL responses also benefitted from IL-33 signaling.
Genealogic artARENA vector backbone relatedness dictates interference by pre-existing immunity and potency in heterologous prime-boost immunization Our observations shown in Figures 3E and 3J suggested that heterologous artARENA prime-boost immunization overruled the immunodominance of vector backbone-directed CTL responses to focus immune responses on vaccine targets. To systematically investigate this hypothesis, we preimmunized mice with LCMV-WT, PICV-WT, CAND-WT, or WT Mopeia virus (MOPV-WT) and studied the animals' ability of responding to artLCMV-E7E6 vaccination 1 month later. MOPV is an Old World mammarenavirus and, thus, has a close genealogic relationship with LCMV, whereas the New World viruses PICV and CAND are only distantly related to LCMV ( Figure 1D). Because of epitope sequence homology between LCMV and MOPV ( Figure 5A), both viruses elicited clearly detectable CTL responses to NP396 whereas the more distantly related PICV-WT and CAND-WT did not ( Figure 5B). Pre-existing immunity to LCMV or MOPV almost completely abrogated E7-specific CTL induction by artLCMV-E7E6, whereas the responses of PICV-WT-or CAND-WT-immune mice were only modestly below those of control mice without prior arenavirus infection ( Figure 5C). This interference by LCMV-WT and MOPV-WT immunity was accompanied by high-frequency NP396-directed responses and a biased E7:NP396 immunodominance hierarchy upon artLCMV-E7E6 vaccination (Figures 5D and 5E). We relied on the immunodominant NP396 epitope as an indicator of backbone crossreactivity, which likely comprised additional epitopes in the four viral backbone proteins NP, GP, L, and Z.
Next, we tested whether these interference and immunodominance hierarchies correlated with the immunogenicity of heterologous artARENA vector prime-boost combinations. To further expand the quiver of artARENA vectors, we generated a reverse genetic system for MOPV and, based thereupon, developed an artMOPV-P1A vector. Cell culture experiments demonstrated attenuated growth of artMOPV-P1A compared with its parental virus ( Figure S7), analogous to the artARENA vectors described in Figures 1E-1G. We compared the ability of artLCMV-P1A to boost P1A-specific CTL responses induced by artPICV-P1A, artCAND-P1A, or artMOPV-P1A in BALB/c mice. Animals undergoing homologous artLCMV-P1A, art-PICV-P1A, or artCAND-P1A prime-boost immunizations served as comparators and, after boost, remained in the 10% range or below ( Figure 5F). Among the heterologous artARENA vector combinations, artPICV-P1A-and artCAND-P1A-primed animals were boosted efficiently by artLCMV-P1A, reaching frequencies in the 20%-30% range. Conversely, P1A-specific CTL responses of artMOPV-P1A-primed and artLCMV-P1A-boosted animals remained below 3%, failing to exceed the responses upon homologous artLCMV-P1A prime-boost. This indicated that the immunogenic benefit of heterologous prime-boost over homologous prime-boost was abolished when the closely related MOPV and LCMV vector backbones were combined. The above hierarchy of heterologous prime-boost combinations correlated inversely with the LCMV NP118-specific CTL responses to prime and boost ( Figure 5G). artPICV-P1A and art-CAND-P1A prime did not induce NP118-specific CTLs above technical background and repressed NP118-directed responses upon artLCMV-P1A boost. Conversely, artMOPV-P1A-primed and artLCMV-P1A-boosted animals had more than 50% NP118-specific CD8 + T cells in peripheral blood with a corresponding P1A:NP118 epitope dominance ratio of less than 0.1 ( Figure 5H). Thus, genealogic vector backbone relatedness and vector backbone-biased CTL responses correlated with inefficient induction of P1A-specific responses.
Interference by vector backbone-specific CTLs rather than by nAbs Next, we investigated the induction and cross-reactive neutralizing activity of artARENA vector-induced antibody responses. Homologous prime-boost immunization with artCAND induced sizeable vector-nAb titers ( Figure 6A). We failed to detect artLCMV-or artPICV-nAbs after homologous prime-boost immunization ( Figure 6A), and neither artLCMV nor artPICV or art-CAND immune sera cross-neutralized any of the other viruses. nAb induction by artCAND, but not artLCMV or artPICV, was in line with differentially dense glycan shields on the respective viruses' envelope proteins. 45 These findings argued against vector-nAbs as a limiting factor in artLCMV-or artPICV-based homologous or heterologous prime-boost immunization. To formally rule out antibody-mediated inhibition, we performed homologous and heterologous prime-boost immunizations in B cell-deficient mice (JHT mice) and WT control mice. E7-specific responses to artPICV-E7E6 and artLCMV-E7E6 prime were indistinguishable in the two strains of mice ( Figure 6B). More importantly, however, JHT and WT mice had a virtually identical response pattern to homologous artLCMV-E7E6 or heterologous artPICV-E7E6 boost, respectively. The superior immunogenicity of heterologous compared with homologous prime-boost despite a lack of antivector antibodies in JHT mice excluded anti-vector antibody responses as a major limitation in homologous artLCMV primeboost immunization ( Figure 6B).
To address whether anti-vector CTL responses, independently of other components of anti-vector immunity, can interfere with artLCMV-based vaccination, we ''preimmunized'' mice with a recombinant vaccinia virus expressing the NP396 epitope as a minigene (VACC-NP396). Control animals were either given vaccinia virus expressing an irrelevant transgene (Vacc-lacZ) as preimmunization or were left unimmunized. As expected, VACC-NP396 preimmunization, but not VACC-lacZ, induced a CTL response to NP396 ( Figure 6C), whereas both viruses triggered CTLs to the immunodominant vaccinia virus backbone epitope B8R 20-27 ( Figure 6D). When subsequently vaccinated with artLCMV-E7E6, the E7-specific CTL responses of VACC-NP396-preimmune animals were 7-to 8-fold lower than those of VACC-lacZ-preimmune mice or controls not previously exposed to vaccinia virus ( Figure 6E). Conversely, the NP396-specific CTL response of VACC-NP396-preimmune mice approached 50% of the circulating CD8 + T cell pool, vastly exceeding the responses of VACC-lacZ-preimmune or vaccinia virus-naive mice ( Figure 6F). Thus, pre-existing immunity to one immunodominant CD8 T cell epitope in the artLCMV backbone was sufficient to repress E7-directed CTL responses upon vaccination with a concomitant shift in E7:NP396 epitope dominance ( Figure 6G).
Heterologous artARENA vector immunotherapy increases TIL numbers and tumor cure rates, resulting in long-term anti-tumor immunity To assess whether the augmented immunogenicity of heterologous artARENA vector prime-boost translated into superior ther-apeutic efficacy, we exploited two transplantable syngeneic mouse tumor models. The P815 mastocytoma cell line is derived from a DBA/2 mouse and expresses the P1A cancer-testis antigen, whereas the C57BL/6-derived TC-1 cell lines serves as a model of HPV16 E7E6-expressing cancer. We implanted P815 and TC-1 tumors subcutaneously into the flank of mice and initiated artPICV or artLCMV vector therapy when tumors were palpable (P815, day 9) or had reached an average critical volume ($100 mm 3 ; TC-1, day 8). artPICV-artLCMV heterologous therapy delivering the respective tumor antigen, P1A or E7E6, was compared with homologous artPICV-artPICV or artLCMV-artLCMV prime-boost, all administered at an interval of 7 days (P815 model) or 10 days (TC-1 model). Homologous artPICV and homologous artLCMV immunotherapy afforded clear tumor volume control compared with untreated control animals (Figures 7A and 7B). In both tumor models, however, the most pronounced and durable effect on tumor volume was seen upon heterologous artPICV-artLCMV therapy. This therapeutic effect depended on the vectorized antigen; a control group of P815 tumor-bearing mice that were given artPICV-GFP-artLCMV-GFP prime-boost, delivering the irrelevant GFP transgene instead of a TAA, did not show a clear therapeutic effect. Homologous prime-boost with TAA-expressing artLCMV or artPICV extended Article ll OPEN ACCESS the survival of tumor-bearing mice, albeit to a lesser extent than heterologous prime-boost, and all mice receiving homologous prime-boost immunization eventually reached humane endpoints ( Figures 7C and 7D). In contrast, 18 percent of mice with P815 tumors and 37.5 percent of animals with TC-1 tumors rejected their respective tumors when undergoing heterologous TAA-vectorizing artPICV-artLCMV immunotherapy (P1A and E7E6 but not GFP), resulting in the animals' survival for more than 125 days (Figures 7C and 7D). When these long-term survivors were re-challenged with the same tumor cells, no tumor growth was recorded, whereas tumor-and therapy-naive control animals progressed rapidly to humane endpoints ( Figures 7E  and 7F). This observation indicated that elimination of established tumors by artPICV-artLCMV immunotherapy resulted in long-term anti-tumor immunity.

DISCUSSION
Elicitation of clinically effective tumor-specific CTL responses remains an important unmet medical need. Here we identify epitope dominance and interference by vector backbonedirected T cell responses as an important hurdle in vectored vaccine delivery. Based on this concept in conjunction with arenaviral genealogy analyses, we developed potent heterologous artARENA-based immunization regimens inducing effective anti-tumor immunity.
Therapeutic cancer vaccination is a particularly demanding field of active immunization. Induction of high-frequency CTL responses against TAAs is often encumbered by central as well as peripheral tolerance mechanisms, limiting the available T cell repertoire and its responsiveness. 72 Additional challenges arise from chronic antigenic exposure, which can lead to functional adaptation or impairment of specific T cell responses. [73][74][75] In this context, potent viral vector systems and their optimal combination are of particular importance. Although poxvirus delivery systems have been evaluated clinically as cancer vaccines for more than 2 decades, 18,76 a wide variety of viral vector systems has entered clinical testing in recent years. These approaches comprise alphavirus vectors, 20 human and sAd-based vectors, 21,22 lentivirus delivery systems, 23 rhabdovirus vectors, 24,25 as well as combinations thereof, attesting to the promise of and broad interest in virally vectored cancer vaccination.
Animal studies have shown that arenavirus vectors are immunogenic when administered intravenously, intradermally, subcutaneously, or intramuscularly, 41,43 and the latter route has also been validated clinically in a human trial. 44 Earlier studies from our lab revealed, however, that the IL-33-ST2 alarmin pathway contributes essentially to the effectiveness of artARENAvectored cancer immunotherapy, 29 and the vector's ability to trigger this pathway correlates with its spread into IL-33-expressing splenic stromal cells. In line with other investigators' work, 77 these findings highlight the importance of vaccine delivery to specialized compartments of secondary lymphoid organs. The intravenous route provides optimal access to these tissues and, therefore, has been used here; it is also exploited in an ongoing clinical trial for artLCMV-based therapy of HPV16-positive head and neck cancer. 49 A discriminating feature of artLCMV and artPICV vector technology is the lack of vector-nAb induction 29,41,43,44 because of the viral glycan shield 45 and globally low arenavirus seroprevalence. [78][79][80][81] The reason for the latter is the natural host range of mammarenaviruses being restricted to rodents, combined with only rare transmission from rodents to humans. Although they do not elicit nAbs against their own glycan-shielded envelope protein, replication-deficient rLCMV vectors elicit potent antibody immunity against vectorized cargo. 29,41,[43][44][45] While future work should investigate the utility of artARENA vector technology for prophylactic antibody induction against infectious diseases, potent CD8 T cell responses position heterologous ar-tARENA vector prime-boost immunization as a promising strategy for therapeutic vaccination in persistent microbial diseases, notably HIV and hepatitis B. 7,82 Our findings highlight anti-vector T cell immunity as a mechanism of interference in artARENA vector homologous primeboost vaccination. For delivery systems such as Ad and poxvirus vectors, which readily elicit potent vector-nAbs, 32,83 the latter are commonly taken as surrogate and supposed main mechanisms of interference by anti-vector immunity. Several observations suggest, however, that pre-existing anti-vector T cells impede responses to Ad and poxvirus vector systems, too. Inefficient responses to MVA-vectored TAAs has been accredited to epitope dominance and competition by vector backbonedirected T cell responses. 84 Similarly, the inhibitory activity of Ad vector-nAbs 83 may have obscured the contribution of additional interference mechanisms. Certain pairs of adenoviral vectors, although serologically distinct, failed to effectively boost each other. 36,37 This indicates that the mere absence of vector-nAbs cannot predict efficient boosting. Conversely, pre-existing Ad-specific T cell immunity is correlated inversely with HIV-specific CTL induction by rAd5 vectors in human vaccine trials. 85 Pre-existing T cell reactivity to Ad5 is found in more than 80% of healthy adults, irrespective of Ad5-specific serostatus. 86 It originates from prior exposure to Ads of unrelated serotypes, targets epitopes in conserved regions of the viral genome, 87 and expands significantly upon rAd5-vectored vaccination. 86 These considerations highlight advantages of viral vector platforms based on virus families such as Arenaviridae, which circulate almost exclusively in the animal kingdom.

Limitations of study
We relied on the transgene:backbone epitope dominance ratio of CD8 T cell responses as an indicator and correlate of backbone-directed T cell interference. Besides inter-clonal competition of transgene-and backbone-specific T cells, 88 an alternative and not mutually exclusive mechanism may consist of accelerated elimination of vector-transduced APCs by pre-existing anti-vector T cells. 89 The present data obtained for mice may imperfectly predict efficacy in humans, but they call for immediate clinical evaluation of heterologous artPICV-artLCMV prime-boost immunization regimens to benefit patients. artPICV-E7E6 and artLCMV-E7E6 are currently entering clinical phase 1 testing as a repeated alternating prime-boost immunization regimen for HPV16-positive head and neck cancer. 49 Irrespective of the limited predictive value of transplantable mouse tumor models, our findings regarding potent CTL induction to self-antigens such as P1A suggest that self-tolerance can be broken efficiently and provide an incentive for our plan of applying artARENA vector technology to a broader range of cancers, most of which do not express viral target antigens. The arenavirus vector platform accommodates transgenes up to $2,000 bp, 29,43 lending itself for delivery of all types of proteinaceous cancer targets, 90 but it remains unknown which ones provide the best clinical efficacy.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
We wish to thank Nadè ge Lagarde, Beatrice Dolder Schlienger, Felix Stemeseder, and Sophie Schulha for excellent technical assistance and Theresa Kleissner, Goran Bekic, Daniel Oeler, and Sonja Feher for diligent implementation of animal studies. This work was supported by the Swiss National Science  All other mouse experiments were performed at the Universities of Basel and Geneva in accordance with the Swiss law for animal protection. Permission was granted by the Veterinä ramt Basel-Stadt and by the Direction gé né rale de la santé , Domaine de l'expé rimentation animale, of the Canton of Geneva, respectively. Animals in experimental groups were sex-and age-matched. In general, adult animals of both genders were used to reduce the number of animals bred for research purposes. P1A-specific immunogenicity assessments and P815 tumor control studies were conducted in female mice. Mice in tumor therapy experiments were assigned to groups in a manner to assure even distribution of tumor volumes between groups at the time of tumor therapy. In accordance with the Swiss law for animal protection mice exhibiting wounds on the tumor or displaying signs of distress (evident namely in lethargy, hunchback, piloerection, emaciation and agonal breathing) were immediately euthanized irrespective of tumor size and diameter. Study sample sizes in animal experiments were chosen based on experience in our labs with respect to group sizes readily revealing biologically significant differences in the experimental models used. The groups were neither randomized nor were experiments conducted in a blinded fashion.
The PICV virulence study in guinea pigs was conducted at Meditox (Czech Republic) and was approved by the Institutional Animal Care and Use Committee (IACUC) and the Committee for Animal Protection of the Ministry of Health of the Czech Republic. Dunkin Hartley guinea pigs were from Charles River, France, and weighed 370 -520 g at the start of the study.
Cell lines BHK-21 cells, HEK293T cells were purchased from ECACC (Clone 13, Cat #85011433), P815 mastocytoma cells (TIB-64), NIH 3T3 and BSC40 cells from ATCC. FreeStyle 293-F suspension culture cells were purchased from Invitrogen/ThermoFisher. LCMV-GPexpressing BHK-21 cells (BHK-21-GP) and 293T-GP cells have previously been described. 41 All cell lines were regularly tested for mycoplasma and were negative. Owing to their origin from renowned international repositories and vendors they were not authenticated.

Viruses, titration and neutralization test
The titration of LCMV, PICV, MOPV and derived vectors by immunofocus assay has been described 29,91 and was performed using NIH 3T3 cells as a substrate, CAND and derived vectors were titrated using HEK293T cells by analogous techniques. For detection of GFP-expressing artPICV and r3PICV infectivity by immunofocus assay we used rat-anti-GFP antibody (Biolegend). 29 To quantify PICV and derived vectors by immunofocus assays, monoclonal antibody (mAb) 17.2.E4-2 95 served as primary antibody. mAb 17.1.C6-9 92 was used for detection of CAND and mAb 2B5 93 for MOPV. LCMV, PICV and derived vectors batches were produced on BHK-21 cells and 293F cells, MOPV and artMOPV-P1A on BHK-21, CAND and derived vectors on 293T cells.
The neutralizing capacity of immune serum was determined by immunofocus reduction assays. 91

Viral virulence testing
Intracranial LCMV infection was administered through the skull and mice developing signs of terminal disease were euthanized in accordance with the Swiss law. The wellbeing of guinea pigs undergoing PICV or artPICV infection was monitored twice daily during the entire study and clinically scored. Moribund animals were sacrificed. Humane endpoints were hypothermia (body temperature < 35 C, determined at two independent monitoring time points) and/or body weight loss S20%.
Tumor implantation and tumor measurement P815 cells (10 6 per mouse) were implanted subcutaneously in the right flank. Tumor growth was assessed three times per week. The longest and the shortest diameter were determined using a caliper. Tumor volumes (mm 3 ) were calculated as ½ (length*width^2). When tumor volumes exceeded 1500 mm 3 or when the longest median tumor diameter exceeded 20 mm, mice were euthanized in accordance with the Swiss law.
TC-1 cells expressing HPV 16 E6 and E7 96 were obtained from Johns Hopkins University. For tumor implantation, 10 5 cells were injected subcutaneously into the flank of C57BL/6 mice and, in accordance with the Austrian law, the experiment was terminated when tumor sizes exceeded 20 mm in any dimension.

Virus engineering, infection and immunization
The reverse genetic engineering of LCMV-WT, r3LCMV and artLCMV vectors using a polymerase I-/ polymerase II-based plasmid system has been described. 39 PICV-based, CAND-based and MOPV-based vectors and the corresponding cDNA-derived WT viruses were generated using analogous expression cassettes and transfection procedures using BHK-21-GP cells as a cell substrate. In brief, we transfected 5x10 5 cells, seeded the day before into an M6 cell culture well, with 0.8 mg of each pol-I-driven S segment expression plasmid, 1 mg of pol-I-driven L segment, 1.4 mg of pol-II-driven L ORF expression plasmid and 0.8 mg of pol-II-driven NP expression plasmid using 12 ml Lipofectamine 2000. Six hours after transfection for the rescue of CAND and derived vectors, Cell Reports Medicine 2, 100209, March 16, 2021 e4 Article ll OPEN ACCESS using the respective single-use chambers in a Immunospot S6 device (C.T.L.). Total numbers of peptide-MHC tetramer-binding CTLs were back calculated. Cytokine profiles after restimulation with overlapping peptide sets spanning the E7 and E6 proteins of HPV16, respectively (JPT) were determined in intracellular cytokine assays as previously described. 31 Samples were measured on BD LSRFortessa flow cytometers and were analyzed using FlowJo software (Becton Dickinson).
For TIL analysis, tumors were dissected and digested with accutase (Sigma Aldrich), Collagenase IV (Worthington), Hyaluronidase (Sigma Aldrich) and DNaseI (Sigma Aldrich) for 60 min at 37 C, followed by red blood cell lysis. Single cell suspensions were filtered using a cell strainer (70 mM).

QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical testing For statistical analysis, GraphPad Prism software (Version 9.0, GraphPad Software) was used unless stated otherwise. Differences between two groups were generally assessed using unpaired two-tailed Student's t tests. To compare one group against multiple other groups we used one-way Analysis Of Variance (ANOVA), followed by Dunnett's post hoc test (Figures 7G-7J). Two-way ANOVA with Sidak's post-test was used to compare multiple cytokine-producing T cell subpopulations of two groups.
Tumor volume curves were compared as described. 104 In brief, the area under the curve (AUC) for each individual animal was calculated using GraphPad Prism software and groups were compared pairwise by two-tailed Mann-Whitney tests. To calculate AUCs for all groups and animals throughout the 41d and 60d periods in Figures 7A and 7B, respectively, animals reaching humane endpoints of tumor volume were assigned either the maximally permitted tumor volume (1500 mm 3 , termination criterion for Figure 7A) or the maximally reached tumor (2815 mm 3 for Figure 7B) for time points after sacrifice. For comparison of long-term survival rates chi-square tests were performed using the GraphPad QuickCalcs online tool.