Nonreplicating synthetic mRNA vaccines: A journey through the European (Journal of Immunology) history

The first worldwide article reporting that injections of synthetic nonreplicating mRNA could be used as a vaccine, which originated from a French team located in Paris, was published in the European Journal of Immunology (EJI) in 1993. It relied on work conducted by several research groups in a handful of countries since the 1960s, which put forward the precise description of eukaryotic mRNA and the method to reproduce this molecule in vitro as well as how to transfect it into mammalian cells. Thereafter, the first industrial development of this technology began in Germany in 2000, with the founding of CureVac, which stemmed from another description of a synthetic mRNA vaccine published in EJI in 2000. The first clinical studies investigating mRNA vaccines in humans were performed as collaboration between CureVac and the University of Tübingen in Germany as early as 2003. Finally, the first worldwide approved mRNA vaccine (an anti‐COVID‐19 vaccine) is based on the mRNA technologies developed by BioNTech since its 2008 foundation in Mainz, Germany, and earlier by the pioneering academic work of its founders. In addition to the past, present, and future of mRNA‐based vaccines, the article aims to present the geographical distribution of the early work, how the development of the technology was implemented by several independent and internationally distributed research teams, as well as the controversies on the optimal way to design or formulate and administer mRNA vaccines

Characterization of the ribonuclease activity on the skin surface

The rapid degradation of ribonucleic acids (RNA) by ubiquitous ribonucleases limits the efficacy of new therapies based on RNA molecules. Therefore, our aim was to characterize the natural ribonuclease activities on the skin and in blood plasma i.e. at sites where many drugs in development are applied. On the skin surfaces of Homo sapiens and Mus musculus we observed dominant pyrimidine-specific ribonuclease activity. This activity is not prevented by a cap structure at the 5'-end of messenger RNA (mRNA) and is not primarily of a 5'- or 3'-exonuclease type. Moreover, the ribonuclease activity on the skin or in blood plasma is not inhibited by chemical modifications introduced at the 2'OH group of cytidine or uridine residues. It is, however, inhibited by the ribonuclease inhibitor RNasin(® )although not by the ribonuclease inhibitor SUPERase· In™. The application of our findings in the field of medical science may result in an improved efficiency of RNA-based therapies that are currently in development

Enhancement of antibody-dependent cellular cytotoxicity is associated with treatment response to extracorporeal photopheresis in Sézary syndrome

Sézary syndrome (SS) is a rare, leukemic type of cutaneous T-cell lymphoma (CTCL), for which extracorporeal photopheresis (ECP) is a first-line therapy. Reliable biomarkers to objectively monitor the response to ECP in patients with SS are missing. We examined the quantitative and qualitative impact of ECP on natural killer (NK) cell activity in SS patients, and especially their functional ability for antibody-dependent cell-mediated cytotoxicity (ADCC). Further, we addressed the question whether the magnitude of the effect on ADCC can be associated with the anti-cancer efficacy of ECP in SS patients. We assessed numbers of NK cells, ADCC activity, and treatment response based on blood tumor staging in a cohort of 13 SS patients (8 women, 5 men) treated with ECP as a first-line therapy. Blood samples were collected before treatment start and after an average of 9 months of uninterrupted ECP treatment. NK cell numbers were reduced in SS patients compared to healthy individuals and showed a tendency of recovery after long-term ECP treatment, independent of the clinical response to treatment. Patients with marginal increase (≤1.5 AU-fold) or lack of increase in ADCC activity failed to respond clinically to treatment, while patients with an increased ADCC activity showed a reduction in blood tumor burden. NK-mediated ADCC is selectively enhanced and might be a mechanism underlying the effect of ECP while in addition it can possibly serve as a reliable biomarker to objectively monitor response to ECP in patients with SS

CD8+ T cells specific for a potential HLA-A*0201 epitope from Chlamydophila pneumoniae are present in the PBMCs from infected patients

Infection with the common pathogen Chlamydophila pneumoniae (Cpn, previously Chlamydia pneumoniae) has a high prevalence in patients suffering from arteriosclerosis and may trigger or contribute to heart disease. In mice, CD8-positive T cells are critical for the eradication of the infection and the development of immune memory against Cpn. Although several H2-class I epitopes have been described, no HLA-class I-associated peptides from Cpn are known. In order to define HLA-A*0201 epitopes from Cpn, we focused on the bacterial heat shock proteins (HSP) 60 and 70 which are known to be recognized by the immune system. Using epitope prediction, peptide binding studies and peptide-specific CTLs from HLA-A2 transgenic mice, we could define a potential HSP-70-derived epitope. The study of PBMCs from Cpn-infected individuals using fluorescent MHC tetramers revealed that some patients have CD8+ T cells capable of recognizing the Cpn HSP-70 HLA-A*0201 epitope. Our studies pave the way to the immunomonitoring of the anti-Cpn CTL immune response present in patients suffering from different diseases potentially linked to Cpn or anti-Cpn immunit

mRNA-Based Anti-TCR CDR3 Tumour Vaccine for T-Cell Lymphoma

Efficient vaccination can be achieved by injections of in vitro transcribed mRNA (ivt mRNA) coding for antigens. This vaccine format is particularly versatile and allows the production of individualised vaccines conferring, T-cell immunity against specific cancer mutations. The CDR3 hypervariable regions of immune receptors (T-cell receptor, TCR or B-cell receptor, BCR) in the context of T- or B-cell leukaemia or lymphoma are targetable and specific sequences, similar to cancer mutations. We evaluated the functionality of an mRNA-based vaccine designed to trigger immunity against TCR CDR3 regions in an EL4 T-lymphoma cell line-derived murine in vivo model. Vaccination against the hypervariable TCR regions proved to be a feasible approach and allowed for protection against T-lymphoma, even though immune escape in terms of TCR downregulation paralleled the therapeutic effect. However, analysis of human cutaneous T-cell lymphoma samples indicated that, as is the case in B-lymphomas, the clonotypic receptor may be a driver mutation and is not downregulated upon treatment. Thus, vaccination against TCR CDR3 regions using customised ivt mRNA is a promising immunotherapy method to be explored for the treatment of patients with T-cell lymphomas

Modified tumour antigen-encoding mRNA facilitates the analysis of naturally occurring and vaccine-induced CD4 and CD8 T cells in cancer patients

The development of effective anti-cancer vaccines requires precise assessment of vaccine-induced immunity. This is often hampered by low ex vivo frequencies of antigen-specific T cells and limited defined epitopes. This study investigates the applicability of modified, in vitro-transcribed mRNA encoding a therapeutically relevant tumour antigen to analyse T cell responses in cancer patients. In this study transfection of antigen presenting cells, by mRNA encoding the tumour antigen NY-ESO-1, was optimised and applied to address spontaneous and vaccine-induced T cell responses in cancer patients. Memory CD8+ T cells from lung cancer patients having detectable humoral immune responses directed towards NY-ESO-1 could be efficiently detected in peripheral blood. Specific T cells utilised a range of different T cell receptors, indicating a polyclonal response. Specific killing of a panel of NY-ESO-1 expressing tumour cell lines indicates recognition restricted to several HLA allelic variants, including a novel HLA-B49 epitope. Using a modified mRNA construct targeting the translated antigen to the secretory pathway, detection of NY-ESO-1-specific CD4+ T cells in patients could be enhanced, which allowed the in-depth characterisation of established T cell clones. Moreover, broad CD8+ and CD4+ T cell responses covering multiple epitopes were detected following mRNA stimulation of patients treated with a recombinant vaccinia/fowlpox NY-ESO-1 vaccine. This approach allows for a precise monitoring of responses to tumour antigens in a setting that addresses the breadth and magnitude of antigen-specific T cell responses, and that is not limited to a particular combination of known epitopes and HLA-restriction

The A to I editing landscape in melanoma and its relation to clinical outcome

RNA editing refers to non-transient RNA modifications that occur after transcription and prior to translation by the ribosomes. RNA editing is more widespread in cancer cells than in non-transformed cells and is associated with tumorigenesis of various cancer tissues. However, RNA editing can also generate neo-antigens that expose tumour cells to host immunosurveillance. Global RNA editing in melanoma and its relevance to clinical outcome currently remain poorly characterized. The present study compared RNA editing as well as gene expression in tumour cell lines from melanoma patients of short or long metastasis-free survival, patients relapsing or not after immuno- and targeted therapy and tumours harbouring BRAF or NRAS mutations. Overall, our results showed that NTRK gene expression can be a marker of resistance to BRAF and MEK inhibition and gives some insights of candidate genes as potential biomarkers. In addition, this study revealed an increase in Adenosine-to-Inosine editing in Alu regions and in non-repetitive regions, including the hyperediting of the MOK and DZIP3 genes in relapsed tumour samples during targeted therapy and of the ZBTB11 gene in NRAS mutated melanoma cells. Therefore, RNA editing could be a promising tool for identifying predictive markers, tumour neoantigens and targetable pathways that could help in preventing relapses during immuno- or targeted therapies

RNA with chemotherapeutic base analogues as a dual-functional anti-cancer drug

Nanoparticles of different sizes formulated with unmodified RNA and Protamine differentially engage Toll-like Receptors (TLRs) and activate innate immune responses in vitro. Here, we report that similar differential immunostimulation that depends on the nanoparticle sizes is induced in vivo in wild type as well as in humanized mice. In addition, we found that the schedule of injections strongly affects the magnitude of the immune response. Immunostimulating 130 nm nanoparticles composed of RNA and Protamine can promote lung metastasis clearance but provides no control of subcutaneous tumors in a CT26 tumor model. We further enhanced the therapeutic capacity of Protamine-RNA nanoparticles by incorporating chemotherapeutic base analogues in the RNA; we coined these immunochemotherapeutic RNAs (icRNAs). Protamine-icRNA nanoparticles were successful at controlling established subcutaneous CT26 and B16 tumors as well as orthotopic glioblastoma. These data indicate that icRNAs are promising cancer therapies, which warrants their further validation for use in the clinic. Keywords: 5FU; Chemotherapy; RNA; immunotherapy; toll like receptor; type I interferon

Vaccines against COVID-19: Priority to mRNA-Based Formulations

As of September 2021, twenty-one anti-COVID-19 vaccines have been approved in the world. Their utilization will expedite an end to the current pandemic. Besides the usual vaccine formats that include inactivated viruses (eight approved vaccines) and protein-based vaccines (four approved vaccines), three new formats have been validated: recombinant adenovirus (six approved vaccines), DNA (one approved vaccine), and messenger RNA (mRNA, two approved vaccines). The latter was the fastest (authorized in 2020 in the EU, the USA, and Switzerland). Most Western countries have reserved or use the protein vaccines, the adenovirus vaccines, and mRNA vaccines. I describe here the different vaccine formats in the context of COVID-19, detail the three formats that are chiefly reserved or used in Europe, Canada, and the USA, and discuss why the mRNA vaccines appear to be the superior format