Preclinical evaluation of a MAGE-A3 vaccination utilizing the oncolytic Maraba virus currently in first-in-human trials.

Multiple immunotherapeutics have been approved for cancer patients, however advanced solid tumors are frequently refractory to treatment. We evaluated the safety and immunogenicity of a vaccination approach with multimodal oncolytic potential in non-human primates (NHP) (Macaca fascicularis). Primates received a replication-deficient adenoviral prime, boosted by the oncolytic Maraba MG1 rhabdovirus. Both vectors expressed the human MAGE-A3. No severe adverse events were observed. Boosting with MG1-MAGEA3 induced an expansion of hMAGE-A3-specific CD4+ and CD8+ T-cells with the latter peaking at remarkable levels and persisting for several months. T-cells reacting against epitopes fully conserved between simian and human MAGE-A3 were identified. Humoral immunity was demonstrated by the detection of circulating MAGE-A3 antibodies. These preclinical data establish the capacity for the Ad:MG1 vaccination to engage multiple effector immune cell populations without causing significant toxicity in outbred NHPs. Clinical investigations utilizing this program for the treatment of MAGE-A3-positive solid malignancies are underway (NCT02285816, NCT02879760).

Serum C-Reactive Protein and Congestive Heart Failure as Significant Predictors of Herpes Zoster Vaccine Response in Elderly Nursing Home Residents.

Background: Elderly long-term care residents often exhibit a myriad of risk factors for immune dysfunction, including chronic inflammation and multiple comorbid conditions, which undoubtedly contribute to their enhanced susceptibility to infection. Hence, understanding the factors required for optimal vaccine responsiveness is critical. Methods: We examined 187 elderly nursing home residents (aged 80-102 years) and 50 community-dwelling seniors (aged 60-75 years) immunized with the live-attenuated varicella-zoster virus (VZV) vaccine. Specifically, we examined whether vaccine responsiveness was associated with serum C-reactive protein (CRP), tumor necrosis factor, interleukin 1ß, 6, and 10, leukocyte telomere length, chronic disease status, and frailty. Results: Elderly participants had significantly higher levels of CRP, tumor necrosis factor, and interleukin 6 and shorter leukocyte telomere length. Vaccine responsiveness was inversely related to the CRP level in elderly participants, but not seniors, and those with congestive heart failure were less likely to achieve a 2-fold response (odds ratio, 0.08). The latter relationship is probably due to immunosenescence, because heart failure was associated with increased senescent CD4+ T cells, and reduced naive and effector and central memory CD8+ T cells. Conclusions: In summary, these data improve our understanding of vaccine responsiveness for those in long-term care, suggesting that certain risk factors are associated with a greater likelihood of vaccine failure.

Immunogenicity of Varicella Vaccine and Immunologic Predictors of Response in a Cohort of Elderly Nursing Home Residents.

BACKGROUND: Little is known about the immunogenicity of live-attenuated Oka/Merck varicella zoster virus (VZV)-containing vaccine (hereafter, "varicella vaccine") in frail nursing homes residents nor about immune phenotypes associated with a response. METHODS: A cohort of 190 frail nursing home residents aged 80-102 years and a cohort of 50 community-dwelling seniors aged 60-75 years (a comparison group) received varicella vaccine. Interferon γ (IFN-γ) enzyme-linked immunospot assays were performed before and 6 weeks after vaccination. Cellular markers of immunosenescence were measured in the nursing home elderly. RESULTS: The average number of IFN-γ spot-forming cells at baseline was significantly lower in the elderly nursing home residents than in the community-dwelling seniors. However, following vaccination, the VZV immune response increased in both cohorts, and no difference was noted in the fold difference of the response between the 2 cohorts. Upon further examination of the elderly nursing home residents, we found that higher frequencies of regulatory T cells and cytomegalovirus-specific CD4+ T cells correlated negatively with the magnitude of VZV-specific responses. CONCLUSIONS: The Oka/Merck varicella vaccine induces VZV immunity in elderly nursing home residents that is similar to that produced in community-dwelling seniors. CLINICAL TRIALS REGISTRATION: NCT01328548.

Designed ankyrin repeat proteins are effective targeting elements for chimeric antigen receptors.

BACKGROUND: Adoptive cell transfer of tumor-specific T lymphocytes (T cells) is proving to be an effective strategy for treating established tumors in cancer patients. One method of generating these cells is accomplished through engineering bulk T cell populations to express chimeric antigen receptors (CARs), which are specific for tumor antigens. Traditionally, these CARs are targeted against tumor antigens using single-chain antibodies (scFv). Here we describe the use of a designed ankyrin repeat protein (DARPin) as the tumor-antigen targeting domain. METHODS: We prepared second generation anti-HER2 CARs that were targeted to the tumor antigen by either a DARPin or scFv. The CARs were engineered into human and murine T cells. We then compared the ability of CARs to trigger cytokine production, degranulation and cytotoxicity. RESULTS: The DARPin CARs displayed reduced surface expression relative to scFv CARs in murine cells but both CARs were expressed equally well on human T cells, suggesting that there may be a processing issue with the murine variants. In both the murine and human systems, the DARPin CARs were found to be highly functional, triggering cytokine and cytotoxic responses that were similar to those triggered by the scFv CARs. CONCLUSIONS: These findings demonstrate the utility of DARPins as CAR-targeting agents and open up an avenue for the generation of CARs with novel antigen binding attributes.

The polyfunctionality of human memory CD8+ T cells elicited by acute and chronic virus infections is not influenced by age.

As humans age, they experience a progressive loss of thymic function and a corresponding shift in the makeup of the circulating CD8+ T cell population from naïve to memory phenotype. These alterations are believed to result in impaired CD8+ T cell responses in older individuals; however, evidence that these global changes impact virus-specific CD8+ T cell immunity in the elderly is lacking. To gain further insight into the functionality of virus-specific CD8+ T cells in older individuals, we interrogated a cohort of individuals who were acutely infected with West Nile virus (WNV) and chronically infected with Epstein Barr virus (EBV) and Cytomegalovirus (CMV). The cohort was stratified into young (<40 yrs), middle-aged (41-59 yrs) and aged (>60 yrs) groups. In the aged cohort, the CD8+ T cell compartment displayed a marked reduction in the frequency of naïve CD8+ T cells and increased frequencies of CD8+ T cells that expressed CD57 and lacked CD28, as previously described. However, we did not observe an influence of age on either the frequency of virus-specific CD8+ T cells within the circulating pool nor their functionality (based on the production of IFNγ, TNFα, IL2, Granzyme B, Perforin and mobilization of CD107a). We did note that CD8+ T cells specific for WNV, CMV or EBV displayed distinct functional profiles, but these differences were unrelated to age. Collectively, these data fail to support the hypothesis that immunosenescence leads to defective CD8+ T cell immunity and suggest that it should be possible to develop CD8+ T cell vaccines to protect aged individuals from infections with novel emerging viruses.

Combined mTOR inhibition and OX40 agonism enhances CD8(+) T cell memory and protective immunity produced by recombinant adenovirus vaccines.

The memory CD8(+) T cell population elicited by immunization with recombinant human adenovirus serotype 5 (rHuAd5) vaccines is composed primarily of effector and effector memory cells (T(EM)) with limited polyfunctionality. In this study, we investigated whether treatment with immunomodulators could enhance and/or redistribute the CD8(+) memory population elicited by rHuAd5. Vaccination in combination with both rapamycin (to modulate differentiation) and an OX40 agonist (to enhance costimulation) increased both the quantity and polyfunctionality of the CD8(+) memory T cell population, with expansion of the T(EM) and memory precursor populations. Furthermore, this intervention enhanced protection against multiple virus challenges. Attenuation of adenovirus transgene expression was required to enable the combination rapamycin + OX40 agonist immunomodulatory treatment to further enhance skewing towards central memory formation, indicating that persistence of antigen expression ultimately limits development of this memory population following rHuAd5 immunization. These results demonstrate that during the expansion phase following adenovirus immunization, the level of mammalian target of rapamycin (mTOR) activity, the amount of costimulation and the duration of antigen availability act together to define the magnitude, phenotype, and functionality of memory CD8(+) T cells. Modulation of these factors can be used to selectively manipulate memory formation.

Novel method for differentiation between Trastuzumab and host adaptive response.

Humoral immune response to human epidermal growth factor receptor 2 (HER-2/neu or ErbB-2) has been detected in sera of breast cancer patients and shown to be an appropriate prognostic marker (Taylor et al., 2007). However, since Trastuzumab (Herceptin) is a widely used monoclonal antibody as cancer therapy agent for tumors over-expressing HER-2, there is a need for an efficient way to detect host-generated antibodies against HER-2 without the confounding effect of Herceptin. Here we describe a screening method developed to decipher between host antibodies against HER-2 and that of Herceptin. By producing a series of truncation mutants within the epitope of Herceptin, we were able to inhibit this binding. We demonstrated also that by a three amino acid substitution (PPF→SSS) we were able to abrogate Herceptin binding while generating a highly conserved HER-2 extracellular domain (ECD). By producing a stable cell line that expresses this mutated form of the human HER-2 ECD, we have a source of this protein to probe patient sera. Our method represents a proof of principle that mutated HER-2 which we constructed could be used to distinguish between a host response against HER-2 and the monoclonal antibody Herceptin targeting the same protein.

CD8+ T-cell expansion and maintenance after recombinant adenovirus immunization rely upon cooperation between hematopoietic and nonhematopoietic antigen-presenting cells.

We have recently reported that CD8(+) T-cell memory maintenance after immunization with recombinant human adenovirus type 5 (rHuAd5) is dependent upon persistent transgene expression beyond the peak of the response. In this report, we have further investigated the location and nature of the cell populations responsible for this sustained response. The draining lymph nodes were found to be important for primary expansion but not for memory maintenance, suggesting that antigen presentation through a nonlymphoid source was required. Using bone marrow chimeric mice, we determined that antigen presentation by nonhematopoietic antigen-presenting cells (APCs) was sufficient for maintenance of CD8(+) T-cell numbers. However, antigen presentation by this mechanism alone yielded a memory population that displayed alterations in phenotype, cytokine production and protective capacity, indicating that antigen presentation through both hematopoietic and nonhematopoietic APCs ultimately defines the memory CD8(+) T-cell response produced by rHuAd5. These results shed new light on the immunobiology of rHuAd5 vectors and provide evidence for a mechanism of CD8(+) T-cell expansion and memory maintenance that relies upon both hematopoietic and nonhematopoietic APCs.

Processing of tumor antigen differentially impacts the development of helper and effector CD4+ T-cell responses.

CD4(+) T cells contribute to the antitumor T-cell response as both effectors that promote tumor rejection and helpers that facilitate the activation of other antitumor effector cells, such as CD8(+) T cells. Maximal engagement of both effector and helper CD4(+) T-cell responses is a desirable attribute of cancer vaccines. We have employed the B16F10 murine melanoma model and a series of recombinant adenovirus (Ad) vaccines expressing mutant forms of the tumor antigen, dopachrome tautomerase, to investigate the relationship between antigen processing and the antitumor CD4(+) T-cell response. Our results have revealed an unexpected dichotomy in the generation of helper and effector CD4(+) T-cell responses where CD4(+) T effector responses are dependent upon protein processing and trafficking, whereas CD4(+) T helper responses are not. The results have important implications for strategies aimed at augmenting antigen immunogenicity by altering intracellular processing and localization.

Persistence of transgene expression influences CD8+ T-cell expansion and maintenance following immunization with recombinant adenovirus.

Previous studies determined that the CD8(+) T-cell response elicited by recombinant adenovirus exhibited a protracted contraction phase that was associated with long-term presentation of antigen. To gain further insight into this process, a doxycycline-regulated adenovirus was constructed to enable controlled extinction of transgene expression in vivo. We investigated the impact of premature termination of transgene expression at various time points (day 3 to day 60) following immunization. When transgene expression was terminated before the maximum response had been attained, overall expansion was attenuated, yielding a small memory population. When transgene expression was terminated between day 13 and day 30, the memory population was not sustained, demonstrating that the early memory population was antigen dependent. Extinction of transgene expression at day 60 had no obvious impact on memory maintenance, indicating that maintenance of the memory population may ultimately become independent of transgene expression. Premature termination of antigen expression had significant but modest effects on the phenotype and cytokine profile of the memory population. These results offer new insights into the mechanisms of memory CD8(+) T-cell maintenance following immunization with a recombinant adenovirus.

Characterizing complex polysera produced by antigen-specific immunization through the use of affinity-selected mimotopes.

BACKGROUND: Antigen-based (as opposed to whole organism) vaccines are actively being pursued for numerous indications. Even though different formulations may produce similar levels of total antigen-specific antibody, the composition of the antibody response can be quite distinct resulting in different levels of therapeutic activity. METHODOLOGY/PRINCIPAL FINDINGS: Using plasmid-based immunization against the proto-oncogene HER-2 as a model, we have demonstrated that affinity-selected epitope mimetics (mimotopes) can provide a defined signature of a polyclonal antibody response. Further, using novel computer algorithms that we have developed, these mimotopes can be used to predict epitope targets. CONCLUSIONS/SIGNIFICANCE: By combining our novel strategy with existing methods of epitope prediction based on physical properties of an individual protein, we believe that this method offers a robust method for characterizing the breadth of epitope-specificity within a specific polyserum. This strategy is useful as a tool for monitoring immunity following vaccination and can also be used to define relevant epitopes for the creation of novel vaccines.

The memory T cell response to West Nile virus in symptomatic humans following natural infection is not influenced by age and is dominated by a restricted set of CD8+ T cell epitopes.

We examined the West Nile virus (WNV)-specific T cell response in a cohort of 52 patients with symptomatic WNV infections, including neuroinvasive and non-invasive disease. Although all virus proteins were shown to contain T cell epitopes, certain proteins, such as E, were more commonly targeted by the T cell response. Most patients exhibited reactivity toward 3-4 individual WNV peptides; however, several patients exhibited reactivity toward >10 individual peptides. The relative hierarchy of T cell reactivities in all patients showed a fixed pattern that was sustained throughout the 12-mo period of the current study. Surprisingly, we did not observe any relationship between age and either the breadth or magnitude of the T cell response following infection. We also did not observe a relationship between disease severity and either the breadth or magnitude of the T cell response. The T cell epitopes were distributed in a non-random fashion across the viral polyprotein and a limited number of epitopes appeared to dominate the CD8(+) T cell response within our cohort. These data provide important new insight into the T cell response against WNV in humans.

On the role of CD4+ T cells in the CD8+ T-cell response elicited by recombinant adenovirus vaccines.

We have investigated the role of CD4(+) T cells in the development of the CD8(+) T-cell response after immunization with recombinant adenovirus (rAd). In the absence of CD4(+) T cells, the "unhelped" CD8(+) T-cell population exhibited a reduction in primary expansion and long-term survival that appeared to be due to inadequate priming of naïve T cells. There were few functional or phenotypic differences between the helped and unhelped CD8(+) T-cell populations with the exception of O-glycosylated CD43, a marker of effector cells, which was augmented on the unhelped CD8(+) T-cell population. In some cases, the unhelped CD8(+) T-cell population exhibited reduced ability to control virus infection; however, this appeared to be a function of the reduced frequency of antigen-specific CD8(+) T cells. Most notably, the unhelped CD8(+) T-cell population exhibited no defect in secondary expansion. These results provide insight into the role of CD4(+) T cells during the primary CD8(+) T-cell response generated by rAd vaccines and identify potential benefits and issues that must be considered when using adenovirus vaccines under conditions where CD4(+) T-cell function may be limiting, such as vaccination of human immunodeficiency virus patients.

The magnitude of the CD8+ T cell response produced by recombinant virus vectors is a function of both the antigen and the vector.

Virus-based recombinant vaccines have proven highly effective at generating protective CD8+ T cell responses. Multiple vector platforms are available, however, little is known about the relative influence of the different vectors on the transgene-specific CD8+ T cell population. To address this question, we compared several characteristics of the CD8+ T cell response elicited by recombinant adenovirus (rAd) and vaccinia virus (rVV). We found that following rAd immunization the transgene-specific CD8+ T cell response peaked around day 12 and was larger and more sustained than the response produced by rVV. In addition, the CD8+ T cell response generated by rAd was directed primarily against the transgene, whereas the CD8+ T cell response produced by rVV principally targeted the vector backbone. In addition, we also observed that transgene selection also impacted on the magnitude of the CD8+ T cell response elicited by both vectors. Despite differences in the magnitude of the anti-transgene CD8+ T cell response, both vectors elicited CD8+ T cell populations with similar cytokine production, functional avidity and cytolytic activity. In addition, plasmid priming prior to immunization with either rAd or rVV only impacted the magnitude of the transgene gene specific CD8+ T cell response. Our study demonstrates that both vector and transgene selection can influence the magnitude of the CD8+ T cell response, but they do not influence functionality.

Semliki forest virus and Kunjin virus RNA replicons elicit comparable cellular immunity but distinct humoral immunity.

RNA replicons offer a number of qualities which make them attractive as vaccination vectors. Both alphavirus and flavivirus replicon vaccines have been investigated in preclinical models yet there has been little direct comparison of the two vector systems. To determine whether differences in the biology of the two vectors influence immunogenicity, we compared two prototypic replicon vectors based on Semliki Forest virus (SFV) (alphavirus) and Kunjin virus (KUN) (flavivirus). Both vectors when delivered as naked RNAs elicited comparable CD8+ T cell responses but the SFV vectors elicited greater humoral responses to an encoded cytoplasmic antigen beta-galactosidase. Studies in MHC class II-deficient mice revealed that neither vector could overcome the dependence of CD4+ T cell help in the development of humoral and cellular responses following immunization. These studies indicate that the distinct biology of the two replicon systems may differentially impact the adaptive immune response and this may need to be considered when designing vaccination strategies.

Use of a murine secreted alkaline phosphatase as a non-immunogenic reporter gene in mice.

BACKGROUND: The development of any vector system as a gene delivery system requires its optimization in vitro and in vivo. Preliminary studies frequently involve the use of a reporter gene, which allows for the rapid and simple assay of vector function through monitoring expression levels of the reporter gene. However, evaluation of vector efficacy can be compromised by immune responses directed against immunogenic reporter proteins. METHODS: We have cloned a murine secreted alkaline phosphatase (mSEAP), and explored its use as a reporter gene in the context of an early region 1 (E1)-deleted adenovirus (Ad) vector. Studies involved characterization of gene expression in vitro and in vivo, and immunological responses after gene delivery to mice. RESULTS: In tissue culture, we show that mSEAP is easily measured quantitatively using a sensitive, commercially available chemiluminescent assay, or visualized directly using histological staining. The level of transgene expression from AdmSEAP was similar to that observed for an Ad vector encoding the human placental secreted alkaline phosphatase (hSEAP). After intravenous administration in mice, AdmSEAP continued to express at high levels for the duration of the experiment (1 month), whereas expression from AdhSEAP declined to background levels over the course of the experiment. Although cytotoxic T-lymphocytes were not detected against either the murine or human SEAP proteins in mice, antibodies were readily detected against the human protein. No antibodies were detected to mSEAP. CONCLUSIONS: Taken together, these data illustrate that mSEAP is a sensitive, non-immunogenic reporter gene for preclinical mouse studies.

Cre levels limit packaging signal excision efficiency in the Cre/loxP helper-dependent adenoviral vector system.

Helper-dependent (HD) adenovirus vectors devoid of all viral coding sequences have a large cloning capacity and provide long-term transgene expression in vivo with negligible toxicity, making them attractive vectors for gene therapy. Currently, the most efficient means of producing HD vectors involves coinfecting 293 cells expressing Cre with the HD vector and a helper virus bearing a packaging signal flanked by loxP sites. Cre-mediated packaging signal excision renders the helper virus genome unpackageable but still able to replicate and provide helper functions for HD vector propagation. Typically, helper virus contamination is < or =1% pre- and < or =0.1% postpurification by CsCl banding. While these contamination levels are low, further reduction is desirable. However, this objective has not been realized since the Cre/loxP system was first developed. This lack of progress is due, at least in part, to our lack of understanding of the origins of the contaminating helper virus, thus rendering its reduction or elimination difficult to achieve. This study was designed to investigate the possible sources of contaminating helper virus persisting during HD vector amplification. The results revealed that Cre is limiting in helper virus-infected Cre-expressing 293 cells, thereby permitting helper viruses to escape packaging signal excision and propagate. The results of this study should provide a foundation for developing rational strategies to further reduce or possibly eliminate the contaminating helper virus.