Virus-based Vectors Research Articles

COVID-19 Vaccine Comparison: How to Choose the Best Suiting Vaccine for Different Needs

Varied measurements have been developed to solve the COVID-19 pandemic, and one of the best ways is vaccines. This paper aims to compare different kinds of vaccines and help people or countries to choose the best suitable vaccine needs based on their different needs. It provides a broader and more detailed analysis of variant vaccines on different levels. In the sequence of technology maturity, inactivated vaccines including CoronaVac, BBIBP-CorV vaccine, and WIBP-CorV vaccine, viral-based vector vaccines including AstraZeneca COVID-19 vaccine and Johnson & Johnson’s COVID-19 vaccine, and mRNA vaccines including Moderna vaccine and Pfizer/BioNTech vaccine have been discussed. There are several important elements of these vaccines, for example, mechanism, effectiveness, and side effects. Different criteria make comparison. For effectiveness, in general, Moderna and Pfizer/BioNTech vaccines could be a better choice. For cost-performance ratio, the AstraZeneca COVID-19 vaccine is the best. As for inoculators’ age and race, old people are suggested to take CoronaVac, and Pfizer/BioNTech vaccine and CoronaVac are suitable for all races. In terms of mutant variant distribution, CoronaVac could be the best choice. Based on safety concerns, inactivated vaccines are better choices. For the developing period, mRNA vaccines win the game. In conclusion, this paper provides further directions for countries seeking the best suitable vaccine for their citizens. After comparing the main vaccines available, we provide a clear view of when countries decide. There would be specific vaccines best suiting specific needs of inoculators and countries.

Hyaluronic acid-binding insulin-like growth factor-1: Creation of a gene encoding a bifunctional fusion protein.

Chondrogenic growth factors are promising therapeutic agents for articular cartilage repair. A persistent impediment to fulfilling this promise is a limited ability to apply and retain the growth factors within the region of cartilage damage that is in need of repair. Current therapies successfully deliver cells and/or matrices, but growth factors are subject to diffusion into the joint space and then loss from the joint. To address this problem, we created a novel gene that encodes a bifunctional fusion protein comprised by a matrix binding domain and a growth factor. The gene encodes the hyaluronic acid binding region of the cartilage matrix molecule, versican, and the chondrogenic growth factor, insulin-like growth factor-1 (IGF-1). We delivered the gene in an adeno-associated virus-based plasmid vector to articular chondrocytes. The cells synthesized and secreted the fusion protein gene product. The fusion protein bound to hyaluronic acid and retained the anabolic and mitogenic actions of IGF-1 on the chondrocytes. This proof-of-concept study suggests that the bifunctional fusion protein, in concert with chondrocytes and a hyaluronic acid-based delivery vehicle, may serve as an intra-articular therapy to help achieve articular cartilage repair.

Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants.

Green fluorescent protein (GFP)-tagged viruses are basic research tools widely applied in studies concerning molecular determinants of disease during virus infection. Here, we described a new generation of genetically stable infectious clones of tomato torrado virus isolate Kra (ToTVpJL-Kra) that could infect Nicotiana benthamiana and Solanum lycopersicum. Importantly, a modified variant of the viral RNA2—with inserted sGFP (forming, together with virus RNA1, into ToTVpJL-KraGFP)—was engineered as well. RNA2 of ToTVpJL-KraGFP was modified by introducing an additional open reading frame (ORF) of sGFP flanked with an amino acid-coding sequence corresponding to the putative virus protease recognition site. Our further analysis revealed that sGFP-tagged ToTV-Kra was successfully passaged by mechanical inoculation and spread systemically in plants. Therefore, the clone might be applied in studying the in vivo cellular, tissue, and organ-level localization of ToTV during infection. By performing whole-plant imaging, followed by fluorescence and confocal microscopy, the presence of the ToTVpJL-KraGFP-derived fluorescence signal was confirmed in infected plants. All this information was verified by sGFP-specific immunoprecipitation and western blot analysis. The molecular biology of the torradovirus-plant interaction is still poorly characterized; therefore, the results obtained here opened up new possibilities for further research. The application of sGFP-tagged virus infectious clones and their development method can be used for analyzing plant-virus interactions in a wide context of plant pathology.

Rabies virus-based COVID-19 vaccine CORAVAX\u2122 induces high levels of neutralizing antibodies against SARS-CoV-2

The recently emerged coronavirus SARS-CoV-2, the causative agent of COVID-19, is rapidly spreading in the world. The exponentially expanding threat of SARS-CoV-2 to global health highlights the urgent need for a vaccine. Herein we show the rapid development of a novel, highly efficient, and safe COVID-19 vaccine using a rabies virus-based vector that has proven to be an efficient vaccine against several emerging infectious diseases. This study reports that both a live and an inactivated rabies virus containing the SARS-CoV-2 spike S1 protein induces potent virus-neutralizing antibodies at much higher levels than seen in the sera of convalescent patients. In summary, the results provided here warrant further development of this safe and established vaccine platform against COVID-19.

Peptide Nanoparticles for Gene Packaging and Intracellular Delivery.

Efficient gene transfer is necessary for advanced biotechnologies ranging from gene therapy to synthetic biology. Peptide nanoparticles provide suitable packaging systems promoting targeted gene expression or silencing. Though these systems have yet to match the transfection efficacy of viruses, they are typically devoid of drawbacks characteristic of virus-based vectors, including insertional mutagenesis, low packaging capacities, and strong immune responses. Given the promise nanoparticle formulations hold for gene delivery, methods of their preparation and accurate analysis of their physicochemical and biological properties become indispensable for progress toward systems that seek to outperform viral vectors. Herein, we report a comprehensive protocol for the preparation and characterization of archetypal peptide nanoparticles resulting from nonspecific and noncovalent complexation with RNA and DNA.

Production of high-titer transmission-defective RNA virus-based episomal vector using tangential flow filtration.

In recent years, viral vector based in vivo gene delivery strategies have achieved a significant success in the treatment of genetic diseases. RNA virus-based episomal vector lacking viral glycoprotein gene (ΔG-REVec) is a nontransmissive gene delivery system that enables long-term gene expression in a variety of cell types in vitro, yet in vivo gene delivery has not been successful due to the difficulty in producing high titer vector. The present study showed that tangential flow filtration (TFF) can be effectively employed to increase the titer of ΔG-REVec. Concentration and diafiltration of ΔG-REVec using TFF significantly increased its titer without loss of infectious activity. Importantly, intracranial administration of high titer vector enabled persistent transgene expression in rodent brain.

Development of Tomato bushy stunt virus-based vectors for fusion and non-fusion expression of heterologous proteins in an alternative host Nicotiana excelsiana.

Plant virus-based expression systems are an alternative expression platform for the production of clinically and industrially useful recombinant proteins. Nonetheless, due to a lack of viral vector with the commercial potentials, it is urgent to design and develop new, versatile, and efficient plant virus vectors. The genome of Tomato bushy stunt virus (TBSV) offers an attractive alternative to being modified as a vector for producing heterologous proteins in plants. Here, we developed a set of novel fusion and non-fusion TBSV-CP replacement vectors, which provide more flexible and efficient tools for expressing proteins of interest in plants. An alternative tobacco plant, Nicotiana excelsiana, was used in this study as a host for newly constructed TBSV vectors because the unwanted necrotic effects were reported on the commonly used Nicotiana benthamiana host associated with expression of TBSV-encoded P19 protein. The data showed that TBSV vectors caused a symptomless infection and overexpressed reporter gene in N. excelsiana leaves, demonstrating that N. excelsiana is an ideal host plant for TBSV-mediated heterologous gene expression. Moreover, a TBSV non-fusion vector, dAUG, shows the similar accumulation level of reporter proteins to that of TMV- and PVX-based vectors in side-by-side comparison and provides more flexible aspects than the previously developed TBSV vectors. Collectively, our newly developed TBSV expression system adds a new member to the family of plant viral expression vectors and meanwhile offers a flexible and highly effective approach for producing proteins of interest in plants. KEY POINTS: • The TBSV-based transient expression system has been significantly improved. • The necrotic effects caused by viral P19 protein were avoided by the usage of N. excelsiana as a host plant. • The expression level of the non-fusion vector was similar to the most effective virus vectors reported so far.

Rapid In-Process Monitoring of Lentiviral Vector Particles by High-Performance Liquid Chromatography

Lentiviral vectors (LVs) are a popular gene delivery tool in cell and gene therapy and they are a primary tool for ex vivo transduction of T cells for expression of chimeric antigen receptor (CAR) in CAR-T cell therapies. Extensive process and product characterization are required in manufacturing virus-based gene vectors to better control batch-to-batch variability. However, it has been an ongoing challenge to make quantitative assessments of LV product because current analytical tools often are low throughput and lack robustness and standardization is still required. This paper presents a high-throughput and robust physico-chemical characterization method that directly assesses total LV particles. With simple sample preparation and fast elution time (6.24 min) of the LV peak in 440 mM NaCl (in 20 mM Tris-HCl [pH 7.5]), this ion exchange high-performance liquid chromatography (IEX-HPLC) method is ideal for routine in-process monitoring to facilitate the development of scalable and robust LV manufacturing processes. Furthermore, this HPLC method is suitable for the analysis of all in-process samples, from crude samples such as LV supernatants to final purified products. The linearity range of the standard curve is 3.13 × 108 to 1.0 × 1010 total particles/mL, and both the intra- and inter-assay variabilities are less than 5%.

Highly efficient DNA-free plant genome editing using virally delivered CRISPR-Cas9.

Genome-editing technologies using CRISPR-Cas nucleases have revolutionized plant science and hold enormous promise in crop improvement. Conventional transgene-mediated CRISPR-Cas reagent delivery methods may be associated with unanticipated genome changes or damage1,2, with prolonged breeding cycles involving foreign DNA segregation and with regulatory restrictions regarding transgenesis3. Therefore, DNA-free delivery has been developed by transfecting preassembled CRISPR-Cas9 ribonucleoproteins into protoplasts4 or in vitro fertilized zygotes5. However, technical difficulties in regeneration from these wall-less cells make impractical a general adaption of these approaches to most crop species. Alternatively, CRISPR-Cas ribonucleoproteins or RNA transcripts have been biolistically bombarded into immature embryo cells or calli to yield highly specific genome editing, albeit at low frequency6-9. Here we report the engineering of a plant negative-strand RNA virus-based vector for DNA-free in planta delivery of the entire CRISPR-Cas9 cassette to achieve single, multiplex mutagenesis and chromosome deletions at high frequency in a model allotetraploid tobacco host. Over 90% of plants regenerated from virus-infected tissues without selection contained targeted mutations, among which up to 57% carried tetra-allelic, inheritable mutations. The viral vector remained stable even after mechanical transmission, and can readily be eliminated from mutated plants during regeneration or after seed setting. Despite high on-target activities, off-target effects, if any, are minimal. Our study provides a convenient, highly efficient and cost-effective approach for CRISPR-Cas9 gene editing in plants through virus infection.

Plant Virus-Derived Vectors: Applications in Agricultural and Medical Biotechnology.

Major advances in our understanding of plant viral genome expression strategies and the interaction of a virus with its host for replication and movement, induction of disease, and resistance responses have been made through the generation of infectious molecules from cloned viral sequences. Autonomously replicating viral vectors derived from infectious clones have been exploited to express foreign genes in plants. Applications of virus-based vectors include the production of human/animal therapeutic proteins in plant cells and the specific study of plant biochemical processes, including those that confer resistance to pathogens. Additionally, virus-induced gene silencing, which is RNA mediated and triggered through homology-dependent RNA degradation mechanisms, has been exploited as an efficient method to study the functions of host genes in plants and to deliver small RNAs to insects. New and exciting strategies for vector engineering, delivery, and applications of plant virus-based vectors are the subject of this review.

Orf Virus-Based Vaccine Vector D1701-V Induces Strong CD8+ T Cell Response against the Transgene but Not against ORFV-Derived Epitopes.

The potency of viral vector-based vaccines depends on their ability to induce strong transgene-specific immune response without triggering anti-vector immunity. Previously, Orf virus (ORFV, Parapoxvirus) strain D1701-V was reported as a novel vector mediating protection against viral infections. The short-lived ORFV-specific immune response and the absence of virus neutralizing antibodies enables repeated immunizations and enhancement of humoral immune responses against the inserted antigens. However, only limited information exists about the D1701-V induced cellular immunity. In this study we employed major histocompatibility complex (MHC) ligandomics and immunogenicity analysis to identify ORFV-specific epitopes. Using liquid chromatography-tandem mass spectrometry we detected 36 ORFV-derived MHC I peptides, originating from various proteins. Stimulated splenocytes from ORFV-immunized mice did not exhibit specific CD8+ T cell responses against the tested peptides. In contrast, immunization with ovalbumin-expressing ORFV recombinant elicited strong SIINFEKL-specific CD8+ T lymphocyte response. In conclusion, our data indicate that cellular immunity to the ORFV vector is negligible, while strong CD8+ T cell response is induced against the inserted transgene. These results further emphasize the ORFV strain D1701-V as an attractive vector for vaccine development. Moreover, the presented experiments describe prerequisites for the selection of T cell epitopes exploitable for generation of ORFV-based vaccines by reverse genetics.

Herpes Simplex Viruses Whose Replication Can Be Deliberately Controlled as Candidate Vaccines.

Over the last few years, we have been evaluating a novel paradigm for immunization using viruses or virus-based vectors. Safety is provided not by attenuation or inactivation of vaccine viruses, but by the introduction into the viral genomes of genetic mechanisms that allow for stringent, deliberate spatial and temporal control of virus replication. The resulting replication-competent controlled viruses (RCCVs) can be activated to undergo one or, if desired, several rounds of efficient replication at the inoculation site, but are nonreplicating in the absence of activation. Extrapolating from observations that attenuated replicating viruses are better immunogens than replication-defective or inactivated viruses, it was hypothesized that RCCVs that replicate with wild-type-like efficiency when activated will be even better immunogens. The vigorous replication of the RCCVs should also render heterologous antigens expressed from them highly immunogenic. RCCVs for administration to skin sites or mucosal membranes were constructed using a virulent wild-type HSV-1 strain as the backbone. The recombinants are activated by a localized heat treatment to the inoculation site in the presence of a small-molecule regulator (SMR). Derivatives expressing influenza virus antigens were also prepared. Immunization/challenge experiments in mouse models revealed that the activated RCCVs induced far better protective immune responses against themselves as well as against the heterologous antigens they express than unactivated RCCVs or a replication-defective HSV-1 strain. Neutralizing antibody and proliferation responses mirrored these findings. We believe that the data obtained so far warrant further research to explore the possibility of developing effective RCCV-based vaccines directed to herpetic diseases and/or diseases caused by other pathogens.

Virus-based vaccines with unique replication mechanisms differentially infect and activate dendritic cells leading to distinctive CD8+ T cell responses

Abstract The precise mechanism by which many virus-based vectors activate immune responses remains unknown. Dendritic cells (DCs) play key roles in priming T cell responses and controlling virus replication, but their functions in generating protective immunity following vaccination with viral vectors are not well understood. We hypothesized that highly immunogenic viral vectors with unique replication mechanisms but identical cell entry pathways differentially infect and activate DCs and promote antigen presentation that leads to efficient induction of distinctive antigen-specific T cell responses. To evaluate differences in replication mechanisms, we utilized a rhabdovirus vector (VSV) and an alphavirus-rhabdovirus hybrid vector (VLV), which replicates like an alphavirus but enters the cell via the VSV glycoprotein. We found that while virus replication promotes CD8+ T cell activation by VLV, replication is absolutely required for VSV-induced responses. DC subtypes were differentially infected in vitro with VSV and VLV, and displayed differences in activation following infection that were dependent on vector replication. Although bone marrow-derived DCs produced type I IFN when infected with the vectors, the mechanism of DC infection and activation was independent of type I IFN receptor signaling. Additionally, the ability of the alphavirus-based vector to generate CD8+ T cells in the absence of replication relied on cDC1 cells that promote antigen cross-presentation. These results highlight the differential activation of DCs following infection with unique viral vectors, and indicate potentially distinct roles of DC subtypes in activating the immune response following immunization.

Optimization of clarification process for viral vectors

Background & Aim Viral-based vector systems such as lentivirus (LV) and adeno-associated virus (AAV) are more widely used and show great potential for delivery of genetic material to the target cells in gene therapy. Downstream processing of lentivirus and adeno-associated virus offers its own unique challenges to generate clinical products of high titer, high potency and high purity.The objective of this work is to identify an efficient clarification step for the separation of viral vectors from the range of impurities found in typical adherent and suspension based viral vector cell culture. These include: host cells, cell debris, aggregates and cell culture media components.The clarification step needs to combine high capacity for impurity removal, high product yield, and ease of scale-up to prepare for downstream operations. Methods, Results & Conclusion Our strategy includes evaluating various depth and membrane filters made up of different organic and inorganic materials for their virus clarification performances. Both AAV and LV processes can be adherent or suspension, each having their own challenges, and in this work, we show an optimized improvement in throughput and recovery for these viral vector feed streams. Thus, making the overall downstream process more efficient, scalable and economical.

Transient expression and purification of β-caryophyllene synthase in Nicotiana benthamiana to produce β-caryophyllene in vitro.

The sesquiterpene β-caryophyllene is an ubiquitous component in many plants that has commercially been used as an aroma in cosmetics and perfumes. Recent studies have shown its potential use as a therapeutic agent and biofuel. Currently, β-caryophyllene is isolated from large amounts of plant material. Molecular farming based on the Nicotiana benthamiana transient expression system may be used for a more sustainable production of β-caryophyllene. In this study, a full-length cDNA of a new duplicated β-caryophyllene synthase from Artemisia annua (AaCPS1) was isolated and functionally characterized. In order to produce β-caryophyllene in vitro, the AaCPS1 was cloned into a plant viral-based vector pEAQ-HT. Subsequently, the plasmid was transferred into the Agrobacterium and agroinfiltrated into N. benthamiana leaves. The AaCPS1 expression was analyzed by quantitative PCR at different time points after agroinfiltration. The highest level of transcripts was observed at 9 days post infiltration (dpi). The AaCPS1 protein was extracted from the leaves at 9 dpi and purified by cobalt–nitrilotriacetate (Co-NTA) affinity chromatography using histidine tag with a yield of 89 mg kg−1 fresh weight of leaves. The protein expression of AaCPS1 was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. AaCPS1 protein uses farnesyl diphosphate (FPP) as a substrate to produce β-caryophyllene. Product identification and determination of the activity of purified AaCPS1 were done by gas chromatography–mass spectrometry (GC–MS). GC–MS results revealed that the AaCPS1 produced maximum 26.5 ± 1 mg of β-caryophyllene per kilogram fresh weight of leaves after assaying with FPP for 6 h. Using AaCPS1 as a proof of concept, we demonstrate that N. benthamiana can be considered as an expression system for production of plant proteins that catalyze the formation of valuable chemicals for industrial applications.

Expanding the genetic code of the human hematopoietic system

The genetic incorporation of noncanonical amino acids (ncAAs) into proteins has been realized in bacteria, yeast, and mammalian cells, and recently, in multicellular organisms including plants and animals. However, the addition of new building blocks to the genetic code of tissues from human origin has not yet been achieved. To this end, we report a self-replicating Epstein-Barr virus-based episomal vector for the long-term encoding of ncAAs in human hematopoietic stem cells and reconstitution of this genetically engineered hematopoietic system in mice.

In vivo biodistribution analysis of transmission competent and defective RNA virus-based episomal vector

RNA virus-based episomal vector (REVec) is an emerging viral vector system that mediates long-term stable gene expression in variety of cell types in vitro. However, little is known about its tissue tropism and persistence of gene expression in vivo. Here, to evaluate the feasibility of REVec for in vivo gene delivery, we conducted biodistribution analysis of transmission competent REVec and transmission defective ΔG-REVec in Lewis rats. Following intracranial administration of REVec, transgene expression was detected in various tissues. In contrast, transgene expression was only observed in the brain after ΔG-REVec administration. Low levels of vector shedding in the feces and blood and of neutralizing antibody in the serum were detected after REVec injection. In the brain, microglia, astrocytes and neurons were susceptible to REVec-mediated transduction. However, the animals administered with REVec, but not with ΔG-REVec showed a significant decrease in body weight compared to mock treated animals. Additionally, CD8 T cell infiltration was observed in the brain of these animals. In summary, we demonstrated that REVec promotes long-term transgene expression in vivo without causing high vector shedding or neutralizing antibody production; however, suggests the need to attenuate vector associated pathogenicity in the future.

Light-Activatable Transfection System Using Hybrid Vectors Composed of Thermosensitive Dendron Lipids and Gold Nanorods.

Background: Gene delivery to target cells is crucially important to establish gene therapy and regenerative medicine. Although various virus-based and synthetic molecule-based gene vectors have been developed to date, selective transfection in a site or a cell level is still challenging. For this study, both light-responsive and temperature-responsive synthetic gene vectors were designed for spatiotemporal control of a transfection system. Methods: 11-Mercaptoundecanoic acid-coated gold nanorods were mixed with polyamidoamine dendron-bearing lipids of two types having amino-terminus or ethoxydiethylene glycol-terminus to obtain hybrid vectors. Hybrid vectors were mixed further with pDNA. Then we investigated their physicochemical properties and transfection efficacy with or without near infrared laser irradiation. Results: Hybrid vectors formed complexes with pDNA and exhibited enhanced photothermal property under near infrared laser irradiation compared with parent gold nanorods. Transfection efficacy of complexes was promoted considerably by brief laser irradiation soon after complex application to the cells. Analysis of intracellular distribution revealed that laser irradiation promoted the adsorption of complexes to the cells and cytosolic release of pDNA, which is derived from the change in surface hydrophobicity of complexes through dehydration of temperature-responsive groups. Conclusions: Hybrid vector is promising as a light-activatable transfection system.

Expression of CD40L by the ALVAC-Simian Immunodeficiency Virus Vector Abrogates T Cell Responses in Macaques.

Immunization with recombinant ALVAC/gp120 alum vaccine provided modest protection from human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) acquisition in humans and macaques. Vaccine-mediated protection was associated with the elicitation of IgG against the envelope V2 loop and of envelope-specific CD4+ T cell responses. We hypothesized that the simultaneous expression of the costimulatory molecule CD40L (CD154) by the ALVAC-HIV vector could increase both protective humoral and cellular responses. We engineered an ALVAC-SIV coexpressing CD40L with SIVmac251 (ALVAC-SIV/CD40L) gag, pol, and env genes. We compared its immunogenicity in macaques with that of a canonical ALVAC-SIV, with both given as a vector-prime/gp120 in alum boost strategy. The ALVAC-SIV/CD40L was superior to the ALVAC-SIV regimen in inducing binding and tier 1 neutralizing antibodies against the gp120. The increase in humoral responses was associated with the expression of the membrane-bound form of the CD40L by CD4+ T cells in lymph nodes. Unexpectedly, the ALVAC-SIV/CD40L vector had a blunting effect on CD4+ Th1 helper responses and instead favored the induction of myeloid-derived suppressor cells, the immune-suppressive interleukin-10 (IL-10) cytokine, and the down-modulatory tryptophan catabolism. Ultimately, this strategy failed to protect macaques from SIV acquisition. Taken together, these results underlie the importance of balanced vaccine-induced activating versus suppressive immune responses in affording protection from HIV.IMPORTANCE CD40-CD40 ligand (CD40L) interaction is crucial for inducing effective cytotoxic and humoral responses against pathogens. Because of its immunomodulatory function, CD40L has been used to enhance immune responses to vaccines, including candidate vaccines for HIV. The only successful vaccine ever tested in humans utilized a strategy combining canarypox virus-based vector (ALVAC) together with an envelope protein (gp120) adjuvanted in alum. This strategy showed limited efficacy in preventing HIV-1/SIV acquisition in humans and macaques. In both species, protection was associated with vaccine-induced antibodies against the HIV envelope and CD4+ T cell responses, including type 1 antiviral responses. In this study, we tested whether augmenting CD40L expression by coexpressing it with the ALVAC vector could increase the protective immune responses. Although coexpression of CD40L did increase humoral responses, it blunted type 1 CD4+ T cell responses against the SIV envelope protein and failed to protect macaques from viral infection.

First Results of Phase I/II Studies Evaluating Viral Vector-Based Heterologous Prime/Boost Immunotherapy Against Predicted HLA Class I Neoantigens Demonstrate CD8 T Cell Responses In Patients with Advanced Cancers

First Results of Phase I/II Studies Evaluating Viral Vector-Based Heterologous Prime/Boost Immunotherapy Against Predicted HLA Class I Neoantigens Demonstrate CD8 T Cell Responses In Patients with Advanced Cancers