Messenger RNA Nanovaccine in Cancer Immunotherapy

During the past 2 years, messenger RNA (mRNA) nanovaccine has shown its remarkable antiviral efficacy, rapid manufacture, and good safety profile for preventing coronavirus infection. Meanwhile, intracellular delivery of mRNA-based cancer vaccine starts to show great potential to elicit antitumor immunity. mRNA encoding tumor antigens, delivery vehicles, and immune adjuvants are the key components of mRNA cancer vaccine. To achieve robust antitumor efficacy, mRNA encoding tumor antigens need to be efficiently delivered and translated in dendritic cells with concurrent innate immune stimulation to promote antigen presentation. Compared with other types of tumor vaccines, mRNA nanovaccine is featured by efficient antigen expression, high potential for rapid development, low-cost manufacture, and safe administration. In this chapter, we mainly focus on the mRNA synthesis, mRNA modification, delivery vectors with immune-stimulating features, and tumor antigen selection and discuss the future direction of mRNA nanovaccine in cancer immunotherapy. © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023.

Nanocarrier vaccine therapeutics for global infectious and chronic diseases

Immunization has the potential to become a viable weapon for the upcoming pandemic and save millions of lives, while also dramatically lowering the high mortality rate brought on by a number of infectious and chronic illnesses. Despite the success of some vaccinations for infectious illnesses, obstacles remain in avoiding and creating fully protective vaccines. Current COVID-19 pandemic highlights need for vaccination platform improvements. Nanomaterials have been created as a possible nanocarrier to elicit a robust immune response against important global morbidity and mortality drivers by encapsulating targeted antigen and functionalizing nanoparticles with particular molecules. In addition to their application in cancer immunotherapy, nanocarriers are currently being included into the development of vaccines against human immunodeficiency virus (HIV), malaria, TB, and influenza. In order to evaluate conventional and next-generation vaccination platforms, this study focuses on the COVID-19 and cancer vaccine as well as the passage and interaction of nanoparticles with immune cells in the lymph node. It also draws attention to the gaps in current and future HIV, TB, malaria, and influenza vaccinations, as well as nanovaccines. The importance of the dose-dependent vaccine in inducing and maintaining neutralizing antibodies after immunization has been discussed in more detail.

Nanovaccines against Viral Infectious Diseases.

Infectious diseases have always been regarded as one of the greatest global threats for the last century. The current ongoing COVID-19 pandemic caused by SARS-CoV-2 is living proof that the world is still threatened by emerging infectious diseases. Morbidity and mortality rates of diseases caused by Coronavirus have inflicted devastating social and economic outcomes. Undoubtedly, vaccination is the most effective method of eradicating infections and infectious diseases that have been eradicated by vaccinations, including Smallpox and Polio. To date, next-generation vaccine candidates with novel platforms are being approved for emergency use, such as the mRNA and viral vectored vaccines against SARS-CoV-2. Nanoparticle based vaccines are the perfect candidates as they demonstrated targeted antigen delivery, improved antigen presentation, and sustained antigen release while providing self-adjuvanting functions to stimulate potent immune responses. In this review, we discussed most of the recent nanovaccines that have found success in immunization and challenge studies in animal models in comparison with their naked vaccine counterparts. Nanovaccines that are currently in clinical trials are also reviewed.

A "trained immunity" inducer-adjuvanted nanovaccine reverses the growth of established tumors in mice.

Innate immune cells are critical in antitumor immune surveillance and the development of antitumor adaptive cellular immunity. Trained innate immune cells demonstrate immune memory-like characteristics, producing more vigorous immune responses to secondary homologous or heterologous stimuli. This study aimed to investigate whether inducing trained immunity is beneficial when using a tumor vaccine to promote antitumor adaptive immune responses. A biphasic delivery system was developed with the trained immunity inducer Muramyl Dipeptide (MDP) and specific tumor antigen human papillomavirus (HPV) E7 peptide encapsulated by poly(lactide-co-glycolide)-acid(PLGA) nanoparticles (NPs), and the NPs along with another trained immunity agonist, ß-glucan, were further embedded in a sodium alginate hydrogel. The nanovaccine formulation demonstrated a depot effect for E7 at the injection site and targeted delivery to the lymph nodes and dendritic cells (DCs). The antigen uptake and maturation of DCs were significantly promoted. A trained immunity phenotype, characterized by increased production of IL-1ß, IL-6, and TNF-α, was induced in vitro and in vivo in response to secondary homologous or heterologous stimulation. Furthermore, prior innate immune training enhanced the antigen-specific INF-γ-expressing immune cell response elicited by subsequent stimulation with the nanovaccine. Immunization with the nanovaccine completely inhibited the growth of TC-1 tumors and even abolished established tumors in mice. Mechanistically, the inclusion of ß-glucan and MDP significantly enhanced the responses of tumor-specific effector adaptive immune cells. The results strongly suggest that the controlled release and targeted delivery of an antigen and trained immunity inducers with an NP/hydrogel biphasic system can elicit robust adaptive immunity, which provides a promising tumor vaccination strategy.

Ionizable polymeric nanocarriers for the codelivery of bi-adjuvant and neoantigens in combination tumor immunotherapy.

Ionizable lipid nanocarriers have made historical contribution to COVID-19 mRNA vaccines. Here, we report ionizable polymeric nanoparticles that co-deliver bi-adjuvant and neoantigen peptides for cancer immunotherapy in combination with immune checkpoint blockade (ICB). Current cancer ICB benefits only a small subset of patients, largely due to a lack of pre-existing target cells and checkpoint targets for ICB, tumor antigenic heterogeneity, and tumor immunosuppression. Therapeutic vaccines hold the potential to enhance ICB therapeutic efficacy by expanding antitumor cell repertoires, upregulating immune checkpoint levels and hence sensitizing ICB, and reducing tumor immunosuppression. Chemically defined peptide vaccines are attractive, but their current therapeutic efficacy has been limited due to 1) poor vaccine delivery to immunomodulatory lymph nodes (LNs) and antigen (Ag)-presenting cells (APCs), 2) poor immunostimulant adjuvant efficacy with restricted target cell subsets in humans, 3) limited adjuvant/Ag codelivery to enhance Ag immunogenicity, and 4) limited ability to overcome tumor antigenic heterogeneity. Here, we developed nanovaccines (NVs) using pH-responsive polymeric micellular nanoparticles (NPs) for the codelivery of bi-adjuvant [Toll-like receptor (TLR) 7/8 agonist R848 and TLR9 agonist CpG] and peptide neoantigens (neoAgs) to draining LNs for efficient Ag presentation in a broad range of APC subsets. These NVs potentiated the immunogenicity of peptide Ags and elicits robust antitumor T cell responses with memory, and remodeled the tumor immune milium with reduced tumor immunosuppression. As a result, NVs significantly enhanced ICB therapeutic efficacy for murine colorectal tumors and orthotopic glioblastoma multiforme (GBM). These results suggest marked potential of bi-adjuvant/neoAg-codelivering NVs for combination cancer immunotherapy.

Self-assembled multiepitope nanovaccine based on NoV P particles induces effective and lasting protection against H3N2 influenza virus.

Current seasonal influenza vaccines confer only limited coverage of virus strains due to the frequent genetic and antigenic variability of influenza virus (IV). Epitope vaccines that accurately target conserved domains provide a promising approach to increase the breadth of protection; however, poor immunogenicity greatly hinders their application. The protruding (P) domain of the norovirus (NoV), which can self-assemble into a 24-mer particle called the NoV P particle, offers an ideal antigen presentation platform. In this study, a multiepitope nanovaccine displaying influenza epitopes (HMN-PP) was constructed based on the NoV P particle nanoplatform. Large amounts of HMN-PP were easily expressed in Escherichia coli in soluble form. Animal experiments showed that the adjuvanted HMN-PP nanovaccine induced epitope-specific antibodies and haemagglutinin (HA)-specific neutralizing antibodies, and the antibodies could persist for at least three months after the last immunization. Furthermore, HMN-PP induced matrix protein 2 extracellular domain (M2e)-specific antibody-dependent cell-mediated cytotoxicity, CD4+ and CD8+ T-cell responses, and a nucleoprotein (NP)-specific cytotoxic T lymphocyte (CTL) response. These results indicated that the combination of a multiepitope vaccine and self-assembled NoV P particles may be an ideal and effective vaccine strategy for highly variable viruses such as IV and SARS-CoV-2. Electronic Supplementary Material: Supplementary material is available in the online version of this article at 10.1007/s12274-023-5395-6.

A new polysaccharide platform constructs self-adjuvant nanovaccines to enhance immune responses.

BACKGROUND: Nanovaccines have shown the promising potential in controlling and eradicating the threat of infectious diseases worldwide. There has been a great need in developing a versatile strategy to conveniently construct diverse types of nanovaccines and induce potent immune responses. To that end, it is critical for obtaining a potent self-adjuvant platform to assemble with different types of antigens into nanovaccines. RESULTS: In this study, we identified a new natural polysaccharide from the rhizomes of Bletilla striata (PRBS), and used this polysaccharide as a platform to construct diverse types of nanovaccines with potent self-adjuvant property. In the construction process of SARS-CoV-2 nanovaccine, PRBS molecules and RBD protein antigens were assembled into ~ 300 nm nanoparticles by hydrogen bond. For HIV nanovaccine, hydrophobic effect dominantly drove the co-assembly between PRBS molecules and Env expression plasmid into ~ 350 nm nanospheres. Importantly, PRBS can potently activate the behaviors and functions of multiple immune cells such as macrophages, B cells and dendritic cells. Depending on PRBS-mediated immune activation, these self-adjuvant nanovaccines can elicit significantly stronger antigen-specific antibody and cellular responses in vivo, in comparison with their corresponding traditional vaccine forms. Moreover, we also revealed the construction models of PRBS-based nanovaccines by analyzing multiple assembly parameters such as bond energy, bond length and interaction sites. CONCLUSIONS: PRBS, a newly-identified natural polysaccharide which can co-assemble with different types of antigens and activate multiple critical immune cells, has presented a great potential as a versatile platform to develop potent self-adjuvant nanovaccines.

Nanovaccine: A Hope to Triumph the Battle Against Novel Coronavirus Disease 2019 (COVID-19)

BACKGROUND: The novel coronavirus 2019 (COVID-19) infection has caused the global emergence of coronavirus in humans during the last 12 months. Till May 11, 2021, the confirmed global COVID-19 cases and deaths reached 158551526 and 3296855, respectively. METHODS: Goblet cells and ciliated cells in the nose act as the initial infection site of SARS-CoV-2. Thus, mucus immunity is important to protect from infection. The outburst of SARS-CoV-2 infection can be halted only when an effective vaccine will be developed. RESULTS: Globally, over 100 different vaccines are under investigation, including DNA vaccines, RNA vaccines, inactivated virus vaccines, adenovirus-based vaccines, recombinant/ subunit protein vaccines, peptide vaccines, and virus-like particles etc. Inactivated virus vaccines and mRNA, and adenovirus-based vaccines have moved fast into clinical trials. CONCLUSION: Vaccines containing spike protein of SARS-CoV as subunit could effectively prevent binding of coronavirus to the host cell and membrane fusion. Thus, spike protein can be used as a major target for subunit vaccine preparation.

A DNA Prime and MVA Boost Strategy Provides a Robust Immunity against Infectious Bronchitis Virus in Chickens.

Infectious bronchitis (IB) is an acute respiratory disease of chickens caused by the avian coronavirus Infectious Bronchitis Virus (IBV). Modified Live Virus (MLV) vaccines used commercially can revert to virulence in the field, recombine with circulating serotypes, and cause tissue damage in vaccinated birds. Previously, we showed that a mucosal adjuvant system, QuilA-loaded Chitosan (QAC) nanoparticles encapsulating plasmid vaccine encoding for IBV nucleocapsid (N), is protective against IBV. Herein, we report a heterologous vaccination strategy against IBV, where QAC-encapsulated plasmid immunization is followed by Modified Vaccinia Ankara (MVA) immunization, both expressing the same IBV-N antigen. This strategy led to the initiation of robust T-cell responses. Birds immunized with the heterologous vaccine strategy had reduced clinical severity and >two-fold reduction in viral burden in lachrymal fluid and tracheal swabs post-challenge compared to priming and boosting with the MVA-vectored vaccine alone. The outcomes of this study indicate that the heterologous vaccine platform is more immunogenic and protective than a homologous MVA prime/boost vaccination strategy.

Lipid-mRNA nanoparticles landscape for cancer therapy.

Intracellular delivery of message RNA (mRNA) technique has ushered in a hopeful era with the successive authorization of two mRNA vaccines for the Coronavirus disease-19 (COVID-19) pandemic. A wide range of clinical studies are proceeding and will be initiated in the foreseeable future to treat and prevent cancers. However, efficient and non-toxic delivery of therapeutic mRNAs maintains the key limited step for their widespread applications in human beings. mRNA delivery systems are in urgent demand to resolve this difficulty. Recently lipid nanoparticles (LNPs) vehicles have prospered as powerful mRNA delivery tools, enabling their potential applications in malignant tumors via cancer immunotherapy and CRISPR/Cas9-based gene editing technique. This review discusses formulation components of mRNA-LNPs, summarizes the latest findings of mRNA cancer therapy, highlights challenges, and offers directions for more effective nanotherapeutics for cancer patients.

Nanotheranostics and its role in diagnosis, treatment and prevention of COVID-19.

Microbe-related, especially viral-related pandemics have currently paralyzed the world and such pathogenesis is expected to rise in the upcoming years. Although tremendous efforts are being made to develop antiviral drugs, very limited progress has been made in this direction. The nanotheranostic approach can be a highly potential rescue to combat this pandemic. Nanoparticles (NPs) due to their high specificity and biofunctionalization ability could be utilized efficiently for prophylaxis, diagnosis and treatment against microbial infections. In this context, titanium oxide, silver, gold NPs, etc. have already been utilized against deadly viruses like influenza, Ebola, HIV, and HBV. The discovery of sophisticated nanovaccines is under investigation and of prime importance to induce reproducible and strong immune responses against difficult pathogens. This review focuses on highlighting the role of various nano-domain materials such as metallic NPs, magnetic NPs, and quantum dots in the biomedical applications to combat the deadly microbial infections. Further, it also discusses the nanovaccines those are already available for various microbial diseases or are in clinical trials. Finally, it gives a perspective on the various nanotechnologies presently employed for efficient diagnosis and therapy against disease causing microbial infections, and how advancement in this field can benefit the health sector remarkably.

A biepitope, adjuvant-free, self-assembled influenza nanovaccine provides cross-protection against H3N2 and H1N1 viruses in mice.

Currently, the incorporation of multiple epitopes into vaccines is more desirable than the incorporation of a single antigen for universal influenza vaccine development. However, epitopes induce poor immune responses. Although the use of adjuvants can overcome this obstacle, it may raise new problems. Effective antigen delivery vehicles that can function as both antigen carriers and intrinsic adjuvants are highly desired for vaccine development. Here, we report a biepitope nanovaccine that provides complete protection in mice against H3N2 virus as well as partial protection against H1N1 virus. This vaccine (3MCD-f) consists of two conserved epitopes (matrix protein 2 ectodomain (M2e) and CDhelix), and these epitopes were presented on the surface of ferritin in a sequential tandem format. Subcutaneous immunization with 3MCD-f in the absence of adjuvant induces robust humoral and cellular immune responses. These results provide a proof of concept for the 3MCD-f nanovaccine that might be an ideal candidate for future influenza pandemics.

Systemic Neutralizing Antibodies and Local Immune Responses Are Critical for the Control of SARS-CoV-2.

Antibody measurements are primarily used to evaluate experimental and approved COVID-19 vaccines, which is unilateral considering our immune responses' complex nature. Previously, we showed that nanoparticle plasmid DNA adjuvant system, QAC, and MVA based vaccines were immunogenic against SARS-CoV-2. Here, we report on the protective efficacy of systemic humoral and mucosal cell-mediated immune responses in transgenic mice models against SARS-CoV-2 following nanoparticle immunization. Parenteral, intramuscular administration of QAC-based plasmid DNA vaccine-encoding SARS-CoV-2 S and N led to the induction of significant serum neutralizing humoral responses, which reduced viral burden in the lungs and prevented viral dissemination to the brain. In contrast, the mucosal, intranasal administration of a heterologous vaccine elicited significant mucosal cell-mediated immune responses in the lungs that limited lung viral replication. The presented results demonstrate that serum neutralizing humoral and local lung T-cell immune responses are critical for the control of SARS-CoV-2 replication.

Self-Assembling Nanovaccine Confers Complete Protection Against Zika Virus Without Causing Antibody-Dependent Enhancement.

The Zika virus (ZIKV) epidemic poses a substantial threat to the public, and the development of safe and effective vaccines is a demanding challenge. In this study, we constructed a kind of self-assembling nanovaccine which confers complete protection against ZIKV infection. The ZIKV envelop protein domain III (zEDIII) was presented on recombinant human heavy chain ferritin (rHF) to form the zEDIII-rHF nanoparticle. Immunization of mice with zEDIII-rHF nanoparticle in the absence of an adjuvant induced robust humoral and cellular immune responses. zEDIII-rHF vaccination conferred complete protection against lethal infection with ZIKV and eliminated pathological symptoms in the brain. Importantly, the zEDIII-rHF nanovaccine induced immune response did not cross-react with dengue virus-2, overcoming the antibody-dependent enhancement (ADE) problem that is a safety concern for ZIKV vaccine development. Our constructed zEDIII-rHF nanovaccine, with superior protective performance and avoidance of ADE, provides an effective and safe vaccine candidate against ZIKV.

A Review of Different Vaccines and Strategies to Combat COVID-19.

In December 2019, an unknown viral infection emerged and quickly spread worldwide, resulting in a global pandemic. This novel virus caused severe pneumonia and acute respiratory distress syndrome caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). It has caused 6.25 millions of deaths worldwide and remains a major concern for health, society, and the economy. As vaccination is one of the most efficient ways to combat this pandemic, different vaccines were developed in a short period. This review article discusses how coronavirus affected the top nations of the world and the vaccines being used for the prevention. Amongst the vaccines, some vaccines have already been approved, and some have been involved in clinical studies. The article also provides insight into different COVID-19 vaccine platforms, their preparation, working, efficacy, and side effects.

A chitosan-mediated inhalable nanovaccine against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with several antigenic variants, has grown into a global challenge, and the rapid establishment of an immune barrier is crucial to achieving long-term control of the virus. This has led to a great demand for easy preparation and scalable vaccines, especially in low-income countries. Here, we present an inhalable nanovaccine comprising chitosan and SARS-CoV-2 spike protein. The chitosan-mediated nanovaccine enabled a strong spike-specific antibody immune response and augmented local mucosal immunity in bronchoalveolar lavage and lungs, which might be capable of protecting the host from infection without systemic toxicity. In addition, the enhanced adaptive immunity stimulated by chitosan showed potential protection against SARS-CoV-2. Furthermore, inhalation of the nanovaccine induced a comparable antibody response compared to intramuscular injection. This inhalable nanovaccine against SARS-CoV-2 offers a convenient and compliant strategy to reduce the use of needles and the need for medical staff. Electronic Supplementary Material: Supplementary material (the immune activation of CS-mediated nanovacccine on BMDCs, cell viability, immune responses in lungs and BALF, serum chemistry and H&E histopathological analysis.) is available in the online version of this article at 10.1007/s12274-021-4012-9.

Peptide-Based Vaccine against SARS-CoV-2: Peptide Antigen Discovery and Screening of Adjuvant Systems.

The SARS-CoV-2 virus has caused a global crisis, resulting in 0.5 billion infections and over 6 million deaths as of March 2022. Fortunately, infection and hospitalization rates were curbed due to the rollout of DNA and mRNA vaccines. However, the efficacy of these vaccines significantly drops a few months post immunization, from 88% down to 47% in the case of the Pfizer BNT162 vaccine. The emergence of variant strains, especially delta and omicron, have also significantly reduced vaccine efficacy. We propose peptide vaccines as a potential solution to address the inadequacies of the current vaccines. Peptide vaccines can be easily modified to target emerging strains, have greater stability, and do not require cold-chain storage. We screened five peptide fragments (B1-B5) derived from the SARS-CoV-2 spike protein to identify neutralizing B-cell peptide antigens. We then investigated adjuvant systems for efficient stimulation of immune responses against the most promising peptide antigens, including liposomal formulations of polyleucine (L10) and polymethylacrylate (PMA), as well as classical adjuvants (CFA and MF59). Immune efficacy of formulations was evaluated using competitive ELISA, pseudovirion neutralization, and live virus neutralization assays. Unfortunately, peptide conjugation to L10 and PMA dramatically altered the secondary structure, resulting in low antibody neutralization efficacy. Of the peptides tested, only B3 administered with CFA or MF59 was highly immunogenic. Thus, a peptide vaccine relying on B3 may provide an attractive alternative to currently marketed vaccines.

Construction of Orthogonal Modular Proteinaceous Nanovaccine Delivery Vectors Based on mSA-Biotin Binding.

Proteinaceous nanovaccine delivery systems have significantly promoted the development of various high-efficiency vaccines. However, the widely used method of coupling the expression of scaffolds and antigens may result in their structural interference with each other. Monovalent streptavidin (mSA) is a short monomer sequence, which has a strong affinity for biotin. Here, we discuss an orthogonal, modular, and highly versatile self-assembled proteinaceous nanoparticle chassis that facilitates combinations with various antigen cargos by using mSA and biotin to produce nanovaccines. We first improved the yield of these nanoparticles by appending a short sugar chain on their surfaces in a constructed host strain. After confirming the strong ability to induce both Th1- and Th2-mediated immune responses based on the plasma cytokine spectrum from immunized mice, we further verified the binding ability of biotinylated nanoparticles to mSA-antigens. These results demonstrate that our biotinylated nanoparticle chassis could load both protein and polysaccharide antigens containing mSA at a high affinity. Our approach thus offers an attractive technology for combining nanoparticles and antigen cargos to generate various high-performance nanovaccines. In particular, the designed mSA connector (mSA containing glycosylation modification sequences) could couple with polysaccharide antigens, providing a new attractive strategy to prepare nanoscale conjugate vaccines.

Nasal Nanovaccines for SARS-CoV-2 to Address COVID-19.

COVID-19 is still prevalent around the globe. Although some SARS-CoV-2 vaccines have been distributed to the population, the shortcomings of vaccines and the continuous emergence of SARS-CoV-2 mutant virus strains are a cause for concern. Thus, it is vital to continue to improve vaccines and vaccine delivery methods. One option is nasal vaccination, which is more convenient than injections and does not require a syringe. Additionally, stronger mucosal immunity is produced under nasal vaccination. The easy accessibility of the intranasal route is more advantageous than injection in the context of the COVID-19 pandemic. Nanoparticles have been proven to be suitable delivery vehicles and adjuvants, and different NPs have different advantages. The shortcomings of the SARS-CoV-2 vaccine may be compensated by selecting or modifying different nanoparticles. It travels along the digestive tract to the intestine, where it is presented by GALT, tissue-resident immune cells, and gastrointestinal lymph nodes. Nasal nanovaccines are easy to use, safe, multifunctional, and can be distributed quickly, demonstrating strong prospects as a vaccination method for SARS-CoV-2, SARS-CoV-2 variants, or SARS-CoV-n.

Vaccine Candidate Double Mutant Variants of Enterotoxigenic Escherichia coli Heat-Stable Toxin.

Heat-stable enterotoxin (ST) producing enterotoxigenic Escherichia coli (ETEC) strains are among the top four enteropathogens associated with moderate-to-severe diarrhea in children under five years in low-to-middle income countries, thus making ST a target for an ETEC vaccine. However, ST must be mutated to abolish its enterotoxicity and to prevent a potential immunological cross-reaction due to its structural resemblance to the human peptides uroguanylin and guanylin. To reduce the risk of eliciting cross-reacting antibodies with our lead STh-A14T toxoid, L9 was chosen as an additional mutational target. A double mutant vaccine candidate immunogen, STh-L9A/A14T, was constructed by conjugation to the synthetic virus-like mi3 nanoparticle using the SpyTag/SpyCatcher technology. This immunogen elicited STh neutralizing antibodies in mice, but with less consistency than STh-A14T peptide control immunogens. Moreover, individual sera from mice immunized with both single and double mutant variants displayed varying levels of unwanted cross-reacting antibodies. The lowest levels of cross-reacting antibodies were observed with STh-L9K/A14T control immunogens, suggesting that it is indeed possible to reduce the risk of eliciting cross-reacting antibodies by mutation. However, mutant-specific antibodies were observed for most double mutant immunogens, demonstrating the delicate balancing act between disrupting cross-reacting epitopes, keeping protective ones, and avoiding the formation of neoepitopes.