mRNA Cancer Vaccines.

mRNA cancer vaccines are a relatively new class of vaccines, which combine the potential of mRNA to encode for almost any protein with an excellent safety profile and a flexible production process. The most straightforward use of mRNA vaccines in oncologic settings is the immunization of patients with mRNA vaccines encoding tumor-associated antigens (TAAs). This is exemplified by the RNActive® technology, which induces balanced humoral and cellular immune responses in animal models and is currently evaluated in several clinical trials for oncologic indications. A second application of mRNA vaccines is the production of personalized vaccines. This is possible because mRNA vaccines are produced by a generic process, which can be used to quickly produce mRNA vaccines targeting patient-specific neoantigens that are identified by analyzing the tumor exome. Apart from being used directly to vaccinate patients, mRNAs can also be used in cellular therapies to transfect patient-derived cells in vitro and infuse the manipulated cells back into the patient. One such application is the transfection of patient-derived dendritic cells (DCs) with mRNAs encoding TAAs, which leads to the presentation of TAA-derived peptides on the DCs and an activation of antigen-specific T cells in vivo. A second application is the transfection of patient-derived T cells with mRNAs encoding chimeric antigen receptors, which allows the T cells to directly recognize a specific antigen expressed on the tumor. In this chapter, we will review preclinical and clinical data for the different approaches.

CD8 T-cell priming upon mRNA vaccination is restricted to bone-marrow-derived antigen-presenting cells and may involve antigen transfer from myocytes.

Self-amplifying mRNAs (SAM(®) ) are a novel class of nucleic acid vaccines, delivered by a non-viral delivery system. They are effective at eliciting potent and protective immune responses and are being developed as a platform technology with potential to be used for a broad range of targets. However, their mechanism of action has not been fully elucidated. To date, no evidence of in vivo transduction of professional antigen-presenting cells (APCs) by SAM vector has been reported, while the antigen expression has been shown to occur mostly in the muscle fibres. Here we show that bone-marrow-derived APCs rather than muscle cells are responsible for induction of MHC class-I restricted CD8 T cells in vivo, but direct transfection of APCs by SAM vectors is not required. Based on all our in vivo and in vitro data we propose that upon SAM vaccination the antigen is expressed within muscle cells and then transferred to APCs, suggesting cross-priming as the prevalent mechanism for priming the CD8 T-cell response by SAM vaccines.

The ISWI chromatin remodeler organizes the hsrω ncRNA-containing omega speckle nuclear compartments.

The complexity in composition and function of the eukaryotic nucleus is achieved through its organization in specialized nuclear compartments. The Drosophila chromatin remodeling ATPase ISWI plays evolutionarily conserved roles in chromatin organization. Interestingly, ISWI genetically interacts with the hsrω gene, encoding multiple non-coding RNAs (ncRNA) essential, among other functions, for the assembly and organization of the omega speckles. The nucleoplasmic omega speckles play important functions in RNA metabolism, in normal and stressed cells, by regulating availability of hnRNPs and some other RNA processing proteins. Chromatin remodelers, as well as nuclear speckles and their associated ncRNAs, are emerging as important components of gene regulatory networks, although their functional connections have remained poorly defined. Here we provide multiple lines of evidence showing that the hsrω ncRNA interacts in vivo and in vitro with ISWI, regulating its ATPase activity. Remarkably, we found that the organization of nucleoplasmic omega speckles depends on ISWI function. Our findings highlight a novel role for chromatin remodelers in organization of nucleoplasmic compartments, providing the first example of interaction between an ATP-dependent chromatin remodeler and a large ncRNA.

Innate Responses to the Former COVID-19 Vaccine Candidate CVnCoV and Their Relation to Reactogenicity and Adaptive Immunogenicity.

Vaccines are highly effective at preventing severe coronavirus disease (COVID-19). With mRNA vaccines, further research is needed to understand the association between immunogenicity and reactogenicity, which is defined as the physical manifestation of an inflammatory response to a vaccination. This study analyzed the immune response and reactogenicity in humans, post immunization, to the former SARS-CoV-2 mRNA investigational vaccine CVnCoV (CV-NCOV-001 and CV-NCOV-002 clinical trials). Immunogenicity was investigated using whole-blood RNA sequencing, serum cytokine levels, and SARS-CoV-2-specific antibodies. The T cell responses in peripheral blood were assessed using intracellular cytokine staining (ICS) and high-dimensional profiling in conjunction with SARS-CoV-2 antigen-specificity testing via mass cytometry. Reactogenicity was graded after participants' first and second doses of CVnCoV using vaccine-related solicited adverse events (AEs). Finally, a Spearman correlation was performed between reactogenicity, humoral immunity, and serum cytokine levels to assess the relationship between reactogenicity and immunogenicity post CVnCoV vaccination. Our findings showed that the gene sets related to innate and inflammatory immune responses were upregulated one day post CVnCoV vaccination, while the gene sets related to adaptive immunity were upregulated predominantly one week after the second dose. The serum levels of IFNα, IFNγ, IP-10, CXCL11, IL-10, and MCP-1 increased transiently, peaking one day post vaccination. CD4+ T cells were induced in all vaccinated participants and low frequencies of CD8+ T cells were detected by ex vivo ICS. Using mass cytometry, SARS-CoV-2 spike-specific CD8+ T cells were induced and were characterized as having an activated effector memory phenotype. Overall, the results demonstrated a positive correlation between vaccine-induced systemic cytokines, reactogenicity, and adaptive immunity, highlighting the importance of the balance between the induction of innate immunity to achieve vaccine efficacy and ensuring low reactogenicity.

Corrigendum to "Safety and immunogenicity of mRNA-LNP COVID-19 vaccine CVnCoV in Latin American adults: A phase 2 randomized study" [Vaccine: X 11 (2022) 100189].

[This corrects the article DOI: 10.1016/j.jvacx.2022.100189.].

Safety and immunogenicity of mRNA-LNP COVID-19 vaccine CVnCoV in Latin American adults: A phase 2 randomized study.

Background: The COVID-19 vaccine candidate CVnCoV comprises sequence-optimized mRNA encoding SARS-CoV-2 S-protein encapsulated in lipid nanoparticles. In this phase 2a study, we assessed reactogenicity and immunogenicity of two or three doses in younger and older adults. Methods: Younger (18-60 years) and older (>60 years) adults were enrolled in two sites in Panama and Peru to receive either 6 or 12 µg doses of CVnCoV or licensed control vaccines 28 days apart; subsets received a 12 µg booster dose on Day 57 or Day 180. Solicited adverse events (AE) were reported for 7 days and unsolicited AEs for 4 weeks after each vaccination, and serious AEs (SAE) throughout the study. Humoral immunogenicity was measured as neutralizing and receptor binding domain (RBD) IgG antibodies and cellular immunogenicity was assessed as CD4+/CD8 + T cell responses. Results: A total of 668 participants were vaccinated (332 aged 18-60 years and 336 aged > 60 years) including 75 who received homologous booster doses. Vaccination was well tolerated with no vaccine-related SAEs. Solicited and unsolicited AEs were mainly mild to moderate and resolved spontaneously. Both age groups demonstrated robust immune responses as neutralizing antibodies or RBD-binding IgG, after two doses, with lower titers in the older age group than the younger adults. Neither group achieved levels observed in human convalescent sera (HCS), but did equal or surpass HCS levels following homologous booster doses. Following CVnCoV vaccination, robust SARS-CoV-2 S-protein-specific CD4 + T-cell responses were observed in both age groups with CD8 + T-cell responses in some individuals, consistent with observations in convalescing COVID-19 patients after natural infection. Conclusions: We confirmed that two 12 µg doses of CVnCoV had an acceptable safety profile, and induced robust immune responses. Marked humoral immune responses to homologous boosters suggest two doses had induced immune memory.

Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines.

mRNA represents a promising new vaccine technology platform with high flexibility in regard to development and production. Here, we demonstrate that vaccines based on sequence optimized, chemically unmodified mRNA formulated in optimized lipid nanoparticles (LNPs) are highly immunogenic and well tolerated in non-human primates (NHPs). Single intramuscular vaccination of NHPs with LNP-formulated mRNAs encoding rabies or influenza antigens induced protective antibody titers, which could be boosted and remained stable during an observation period of up to 1 year. First mechanistic insights into the mode of action of the LNP-formulated mRNA vaccines demonstrated a strong activation of the innate immune response at the injection site and in the draining lymph nodes (dLNs). Activation of the innate immune system was reflected by a transient induction of pro-inflammatory cytokines and chemokines and activation of the majority of immune cells in the dLNs. Notably, our data demonstrate that mRNA vaccines can compete with licensed vaccines based on inactivated virus or are even superior in respect of functional antibody and T cell responses. Importantly, we show that the developed LNP-formulated mRNA vaccines can be used as a vaccination platform allowing multiple, sequential vaccinations against different pathogens. These results provide strong evidence that the mRNA technology is a valid approach for the development of effective prophylactic vaccines to prevent infectious diseases.

The miR-155-PU.1 axis acts on Pax5 to enable efficient terminal B cell differentiation.

A single microRNA (miRNA) can regulate the expression of many genes, though the level of repression imparted on any given target is generally low. How then is the selective pressure for a single miRNA/target interaction maintained across long evolutionary distances? We addressed this problem by disrupting in vivo the interaction between miR-155 and PU.1 in mice. Remarkably, this interaction proved to be key to promoting optimal T cell-dependent B cell responses, a previously unrecognized role for PU.1. Mechanistically, miR-155 inhibits PU.1 expression, leading to Pax5 down-regulation and the initiation of the plasma cell differentiation pathway. Additional PU.1 targets include a network of genes whose products are involved in adhesion, with direct links to B-T cell interactions. We conclude that the evolutionary adaptive selection of the miR-155-PU.1 interaction is exercised through the effectiveness of terminal B cell differentiation.