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Coronavirus disease 2019 (COVID-19) mRNA vaccines that have contributed to controlling the SARS-CoV-2 pandemic induce specific serum antibodies, which correlate with protection. However, the neutralizing capacity of antibodies for emerging SARS-CoV-2 variants is altered. Suboptimal antibody responses are observed in patients with humoral immunodeficiency diseases, ongoing B cell depletion therapy, and aging. Common experimental mouse models with altered B cell compartments, such as B cell depletion or deficiency, do not fully recapitulate scenarios of declining or suboptimal antibody levels as observed in humans. We report on SARS-CoV-2 immunity in a transgenic mouse model with restricted virus-specific antibodies. Vaccination of C57BL/6-Tg(IghelMD4)4Ccg/J mice with unmodified or N1mΨ-modified mRNA encoding for ancestral spike (S) protein and subsequent challenge with mouse-adapted SARS-CoV-2 provided insights into antibody-independent immunity and the impact of antibody titers on mucosal immunity. Protection against fatal disease was independent of seroconversion following mRNA vaccination, suggesting that virus-specific T cells can compensate for suboptimal antibody levels. In contrast, mRNA-induced IgG in the nasal conchae limited the local viral load and disease progression. Our results indicate that parenteral mRNA immunization can elicit nasal IgG antibodies that effectively suppress local viral replication, highlighting the potential of vaccines in controlling SARS-CoV-2 transmission and epidemiology., Competing Interests: J.G., K.K., N.R., and S.R. are employed by CureVac SE, which develops SARS-CoV-2 vaccines. The Friedrich-Loeffler-Institut receives funding for SARS-CoV-2 vaccine research by CureVac SE (Tübungen, Germany) and the RocketVax AG (Basel, Switzerland). M.B., D.H., and L.U. are part of a patent application for SARS-CoV-2 vaccines., (© 2024 The Author(s).)

Human rotavirus (HRV) is still a leading cause of severe dehydrating gastroenteritis globally, particularly in infants and children. Previously, we demonstrated the immunogenicity of mRNA-based HRV vaccine candidates expressing the viral spike protein VP8* in rodent models. In the present study, we assessed the immunogenicity and protective efficacy of two mRNA-based HRV trivalent vaccine candidates, encoding VP8* of the genotypes P[8], P[6], or P[4], in the gnotobiotic (Gn) pig model of Wa (G1P[8]) HRV infection and diarrhea. Vaccines either encoded VP8* alone fused to the universal T-cell epitope P2 (P2-VP8*) or expressed P2-VP8* as a fusion protein with lumazine synthase (LS-P2-VP8*) to allow the formation and secretion of protein particles that present VP8* on their surface. Gn pigs were randomly assigned into groups and immunized three times with either P2-VP8* (30 µg) or LS-P2-VP8* (30 µg or 12 µg). A trivalent alum-adjuvanted P2-VP8* protein vaccine or an LNP-formulated irrelevant mRNA vaccine served as the positive and negative control, respectively. Upon challenge with virulent Wa HRV, a significantly shortened duration and decreased severity of diarrhea and significant protection from virus shedding was induced by both mRNA vaccine candidates compared to the negative control. Both LS-P2-VP8* doses induced significantly higher VP8*-specific IgG antibody titers in the serum after immunizations than the negative as well as the protein control. The P[8] VP8*-specific IgG antibody-secreting cells in the ileum, spleen, and blood seven days post-challenge, as well as VP8*-specific IFN-γ-producing T-cell numbers increased in all three mRNA-vaccinated pig groups compared to the negative control. Overall, there was a clear tendency towards improved responses in LS-P2-VP8* compared to the P2-VP8*mRNA vaccine. The demonstrated strong humoral immune responses, priming for effector T cells, and the significant reduction of viral shedding and duration of diarrhea in Gn pigs provide a promising proof of concept and may provide guidance for the further development of mRNA-based rotavirus vaccines.

Malaria transmission-blocking vaccines (TBV) are designed to inhibit the sexual stage development of the parasite in the mosquito host and can play a significant role in achieving the goal of malaria elimination. Preclinical and clinical studies using protein-protein conjugates of leading TBV antigens Pfs25 and Pfs230 domain 1 (Pfs230D1) have demonstrated the feasibility of TBV. Nevertheless, other promising vaccine platforms for TBV remain underexplored. The recent success of mRNA vaccines revealed the potential of this technology for infectious diseases. We explored the mRNA platform for TBV development. mRNA constructs of Pfs25 and Pfs230D1 variously incorporating signal peptides (SP), GPI anchor, and Trans Membrane (TM) domain were assessed in vitro for antigen expression, and selected constructs were evaluated in mice. Only mRNA constructs with GPI anchor or TM domain that resulted in high cell surface expression of the antigens yielded strong immune responses in mice. These mRNA constructs generated higher transmission-reducing functional activity versus the corresponding alum-adjuvanted protein-protein conjugates used as comparators. Pfs25 mRNA with GPI anchor or TM maintained >99% transmission reducing activity through 126 days, the duration of the study, demonstrating the potential of mRNA platform for TBV., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Despite the availability of live-attenuated oral vaccines, rotavirus remains a major cause of severe childhood diarrhea worldwide. Due to the growing demand for parenteral rotavirus vaccines, we developed mRNA-based vaccine candidates targeting the viral spike protein VP8*. Our monomeric P2 (universal T cell epitope)-VP8* mRNA design is equivalent to a protein vaccine currently in clinical development, while LS (lumazine synthase)-P2-VP8* was designed to form nanoparticles. Cyro-electron microscopy and western blotting-based data presented here suggest that proteins derived from LS-P2-VP8* mRNA are secreted in vitro and self-assemble into 60-mer nanoparticles displaying VP8*. mRNA encoded VP8* was immunogenic in rodents and introduced both humoral and cellular responses. LS-P2-VP8* induced superior humoral responses to P2-VP8* in guinea pigs, both as monovalent and trivalent vaccines, with encouraging responses detected against the most prevalent P genotypes. Overall, our data provide evidence that trivalent LS-P2-VP8* represents a promising mRNA-based next-generation rotavirus vaccine candidate., (© 2023. The Author(s).)

Knowledge about early immunity to SARS-CoV-2 variants of concern mainly comes from the analysis of human blood. Such data provide limited information about host responses at the site of infection and largely miss the initial events. To gain insights into compartmentalization and the early dynamics of host responses to different SARS-CoV-2 variants, we utilized human angiotensin converting enzyme 2 (hACE2) transgenic mice and tracked immune changes during the first days after infection by RNAseq, multiplex assays, and flow cytometry. Viral challenge infection led to divergent viral loads in the lungs, distinct inflammatory patterns, and innate immune cell accumulation in response to ancestral SARS-CoV-2, Beta (B.1.351) and Delta (B.1.617.2) variant of concern (VOC). Compared to other SARS-CoV-2 variants, infection with Beta (B.1.351) VOC spread promptly to the lungs, leading to increased inflammatory responses. SARS-CoV-2-specific antibodies and T cells developed within the first 7 days postinfection and were required to reduce viral spread and replication. Our studies show that VOCs differentially trigger transcriptional profiles and inflammation. This information contributes to the basic understanding of immune responses immediately postexposure to SARS-CoV-2 and is relevant for developing pan-VOC interventions including prophylactic vaccines., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Licensed rabies virus vaccines based on whole inactivated virus are effective in humans. However, there is a lack of detailed investigations of the elicited immune response, and whether responses can be improved using novel vaccine platforms. Here we show that two doses of a lipid nanoparticle-formulated unmodified mRNA vaccine encoding the rabies virus glycoprotein (RABV-G) induces higher levels of RABV-G specific plasmablasts and T cells in blood, and plasma cells in the bone marrow compared to two doses of Rabipur in non-human primates. The mRNA vaccine also generates higher RABV-G binding and neutralizing antibody titers than Rabipur, while the degree of somatic hypermutation and clonal diversity of the response are similar for the two vaccines. The higher overall antibody titers induced by the mRNA vaccine translates into improved cross-neutralization of related lyssavirus strains, suggesting that this platform has potential for the development of a broadly protective vaccine against these viruses., (© 2023. The Author(s).)

Despite the success of the widely used attenuated yellow fever (YF) vaccine, its global supply remains a substantial barrier to implementing vaccination campaigns in endemic regions and combating emerging epidemics. In A129 mice and rhesus macaques, we evaluated the immunogenicity and protective activity of messenger RNA (mRNA) vaccine candidates encapsulated in lipid nanoparticles, expressing the pre-membrane and envelope proteins or the non-structural protein 1 of YF virus. Vaccine constructs induced humoral and cell-mediated immune responses in mice, resulting in protection against lethal YF virus infection after passive administration of serum or splenocytes from vaccinated mice. Vaccination of macaques induced sustained high humoral and cellular immune responses for at least 5 months after the second dose. Our data demonstrate that these mRNA vaccine candidates can be considered an attractive addition to the licensed YF vaccine supply based on the induction of functional antibodies correlating with protection and T-cell responses; they could alleviate the limited supply of current YF vaccines, mitigating future YF epidemics., (© 2023. The Author(s).)

Combining optimized spike (S) protein-encoding mRNA vaccines to target multiple SARS-CoV-2 variants could improve control of the COVID-19 pandemic. We compare monovalent and bivalent mRNA vaccines encoding B.1.351 (Beta) and/or B.1.617.2 (Delta) SARS-CoV-2 S-protein in a transgenic mouse and a Wistar rat model. The blended low-dose bivalent mRNA vaccine contains half the mRNA of each respective monovalent vaccine, but induces comparable neutralizing antibody titres, enrichment of lung-resident memory CD8 + T cells, antigen-specific CD4 + and CD8 + responses, and protects transgenic female mice from SARS-CoV-2 lethality. The bivalent mRNA vaccine significantly reduces viral replication in both Beta- and Delta-challenged mice. Sera from bivalent mRNA vaccine immunized female Wistar rats also contain neutralizing antibodies against the B.1.1.529 (Omicron BA.1 and BA.5) variants. These data suggest that low-dose and fit-for-purpose multivalent mRNA vaccines encoding distinct S-proteins are feasible approaches for extending the coverage of vaccines for emerging and co-circulating SARS-CoV-2 variants., (© 2023. The Author(s).)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron and its subvariants (BA.2, BA.4, BA.5) represented the most commonly circulating variants of concern (VOC) in the coronavirus disease 2019 (COVID-19) pandemic in 2022. Despite high vaccination rates with approved SARS-CoV-2 vaccines encoding the ancestral spike (S) protein, these Omicron subvariants have collectively resulted in increased viral transmission and disease incidence. This necessitates the development and characterization of vaccines incorporating later emerging S proteins to enhance protection against VOC. In this context, bivalent vaccine formulations may induce broad protection against VOC and potential future SARS-CoV-2 variants. Here, we report preclinical data for a lipid nanoparticle (LNP)-formulated RNActive ® N1-methylpseudouridine (N1mΨ) modified mRNA vaccine (CV0501) based on our second-generation SARS-CoV-2 vaccine CV2CoV, encoding the S protein of Omicron BA.1. The immunogenicity of CV0501, alone or in combination with a corresponding vaccine encoding the ancestral S protein (ancestral N1mΨ), was first measured in dose-response and booster immunization studies performed in Wistar rats. Both monovalent CV0501 and bivalent CV0501/ancestral N1mΨ immunization induced robust neutralizing antibody titers against the BA.1, BA.2 and BA.5 Omicron subvariants, in addition to other SARS-CoV-2 variants in a booster immunization study. The protective efficacy of monovalent CV0501 against live SARS-CoV-2 BA.2 infection was then assessed in hamsters. Monovalent CV0501 significantly reduced SARS-CoV-2 BA.2 viral loads in the airways, demonstrating protection induced by CV0501 vaccination. CV0501 has now advanced into human Phase 1 clinical trials (ClinicalTrials.gov Identifier: NCT05477186).

A third vaccine dose is often required to achieve potent, long-lasting immune responses. We investigated the effect of three 8-μg doses of CVnCoV, CureVac's severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidate containing sequence-optimized unmodified mRNA encoding the spike (S) glycoprotein, administered at 0, 4, and 28 weeks, on immune responses in rhesus macaques. After the third dose, S-specific binding and neutralizing antibodies increased 50-fold compared with post-dose 2 levels, with increased responses also evident in the lower airways and against the SARS-CoV-2 B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) variants. Enhanced binding affinity of serum antibodies after the third dose correlated with higher somatic hypermutation in S-specific B cells, corresponding with improved binding properties of monoclonal antibodies expressed from isolated B cells. Administration of low-dose mRNA led to fewer cells expressing antigen in vivo at the injection site and in the draining lymph nodes compared with a 10-fold higher dose, possibly reducing engagement of precursor cells with the antigen and resulting in the suboptimal response observed after two-dose vaccination schedules in phase IIb/III clinical trials of CVnCoV. However, when immune memory is established, a third dose efficiently boosts the immunological responses and improves antibody affinity and breadth., Competing Interests: S.O.M., E.J., K.S., S.R., and B.P. are employees of CureVac AG., (© 2022 The Authors.)

More than two years after the emergence of SARS-CoV-2, 33 COVID-19 vaccines, based on different platforms, have been approved in 197 countries. Novel variants that are less efficiently neutralised by antibodies raised against ancestral SARS-CoV-2 are circulating, highlighting the need to adapt vaccination strategies. Here, we compare the immunogenicity of a first-generation mRNA vaccine candidate, CVnCoV, with a second-generation mRNA vaccine candidate, CV2CoV, in rats. Higher levels of spike (S) protein expression were observed in cell culture with the CV2CoV mRNA than with the CVnCoV mRNA. Vaccination with CV2CoV also induced higher titres of virus neutralising antibodies with accelerated kinetics in rats compared with CVnCoV. Significant cross-neutralisation of the SARS-CoV-2 variants, Alpha (B.1.1.7), Beta (B.1.351), and the 'mink' variant (B1.1.298) that were circulating at the time in early 2021 were also demonstrated. In addition, CV2CoV induced higher levels of antibodies at lower doses than CVnCoV, suggesting that dose-sparing could be possible with the next-generation SARS-CoV-2 vaccine, which could improve worldwide vaccine supply.

mRNA vaccines can be developed and produced quickly, making them prime candidates for immediate outbreak responses. Furthermore, clinical trials have demonstrated rapid protection following mRNA vaccination. Thus, we sought to investigate how quickly mRNA vaccines elicit antibody responses compared to other vaccine modalities. We first compared the immune kinetics of mRNA and DNA vaccines expressing SARS-CoV-2 spike in mice. We observed rapid induction of antigen-specific binding and neutralizing antibodies by day 5 following mRNA (4 µg/mouse), but not DNA (50 µg/mouse), immunization. Comparing innate responses hours post immunization, the mRNA vaccine induced increased levels of IL-5, IL-6, and MCP-1 cytokines which maybe promoting humoral responses downstream. We then evaluated the immune kinetics of an HIV-1 mRNA vaccine in comparison to DNA, protein, and rhesus adenovirus 52 (RhAd52) vaccines of the same HIV-1 envelope antigen in mice. Again, induction of envelope-specific antibodies was observed by day 5 following mRNA vaccination, whereas antibodies were detected by day 7-14 following DNA, protein, and RhAd52 vaccination. Thus, eliciting rapid humoral immunity may be a unique and advantageous property of mRNA vaccines for controlling infectious disease outbreaks., (© 2022. The Author(s).)

Immunogenicity of HIV-1 mRNA vaccine regimens was analyzed in a non-human primate animal model. Rhesus macaques immunized with mRNA in lipid nanoparticle (mRNA/LNP) formulation expressing HIV-1 Gag and Gag conserved regions (CE) as immunogens developed robust, durable antibody responses but low adaptive T-cell responses. Augmentation of the dose resulted in modest increases in vaccine-induced cellular immunity, with no difference in humoral responses. The gag mRNA/lipid nanoparticle (LNP) vaccine provided suboptimal priming of T cell responses for a heterologous DNA booster vaccination regimen. In contrast, a single immunization with gag mRNA/LNP efficiently boosted both humoral and cellular responses in macaques previously primed by a gag DNA-based vaccine. These anamnestic cellular responses were mediated by activated CD8 + T cells with a phenotype of differentiated T-bet + cytotoxic memory T lymphocytes. The heterologous prime/boost regimens combining DNA and mRNA/LNP vaccine modalities maximized vaccine-induced cellular and humoral immune responses. Analysis of cytokine responses revealed a transient systemic signature characterized by the release of type I interferon, IL-15 and IFN-related chemokines. The pro-inflammatory status induced by the mRNA/LNP vaccine was also characterized by IL-23 and IL-6, concomitant with the release of IL-17 family of cytokines. Overall, the strong boost of cellular and humoral immunity induced by the mRNA/LNP vaccine suggests that it could be useful as a prophylactic vaccine in heterologous prime/boost modality and in immune therapeutic interventions against HIV infection or other chronic human diseases., Competing Interests: Authors JM, GP, BP and BF report issued patents of relevance to this work. Author MAn was employed by Leidos Biomedical Research, Inc. Authors JG, BP, LO, EL are employed by CureVac AG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Valentin, Bergamaschi, Rosati, Angel, Burns, Agarwal, Gergen, Petsch, Oostvogels, Loeliger, Chew, Deeks, Mullins, Pavlakis and Felber.)

In the past 20 years, the mRNA vaccine technology has evolved from the first proof of concept to the first licensed vaccine against emerging pandemics such as SARS-CoV-2. Two mRNA vaccines targeting SARS-CoV-2 have received emergency use authorization by US FDA, conditional marketing authorization by EMA, as well as multiple additional national regulatory authorities. The simple composition of an mRNA encoding the antigen formulated in a lipid nanoparticle enables a fast adaptation to new emerging pathogens. This can speed up vaccine development in pandemics from antigen and sequence selection to clinical trial to only a few months. mRNA vaccines are well tolerated and efficacious in animal models for multiple pathogens and will further contribute to the development of vaccines for other unaddressed diseases. Here, we give an overview of the mRNA vaccine design and factors for further optimization of this new promising technology and discuss current knowledge on the mode of action of mRNA vaccines interacting with the innate and adaptive immune system., (© 2020. Springer Nature Switzerland AG.)

The CVnCoV (CureVac) mRNA vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was recently evaluated in a phase 2b/3 efficacy trial in humans 1 . CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression. Here we report the results of a head-to-head comparison of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in non-human primates. We immunized 18 cynomolgus macaques with two doses of 12 μg lipid nanoparticle-formulated CVnCoV or CV2CoV or with sham (n = 6 per group). Compared with CVnCoV, CV2CoV induced substantially higher titres of binding and neutralizing antibodies, memory B cell responses and T cell responses as well as more potent neutralizing antibody responses against SARS-CoV-2 variants, including the Delta variant. Moreover, CV2CoV was found to be comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques. Although CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded more robust protection with markedly lower viral loads in the upper and lower respiratory tracts. Binding and neutralizing antibody titres were correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates., (© 2021. The Author(s).)

Zika virus (ZIKV), a mosquito-borne flavivirus, has recently triggered global concern due to severe health complications. In 2015, a large ZIKV outbreak occurred in the Americas and established a link between ZIKV and microcephaly in newborn babies, spontaneous abortion, persistent viremia, and Guillain-Barré syndrome. While antivirals are being developed and prevention strategies focus on vector control, a safe and effective Zika vaccine remains unavailable. Messenger RNA (mRNA) vaccine technology has arisen as a flexible, simplified, and fast vaccine production platform. Here, we report on an mRNA vaccine candidate that encodes the pre-membrane and envelope (prM-E) glycoproteins of ZIKV strain Brazil SPH2015 and is encapsulated in lipid nanoparticles (LNPs). Our ZIKV prM-E mRNA-LNP vaccine candidate induced antibody responses that protected in AG129 mice deficient in interferon (IFN) alpha/beta/gamma (IFN-α/β/γ) receptors. Notably, a single administration of ZIKV prM-E mRNA-LNP protected against a lethal dose of ZIKV, while a two-dose strategy induced strong protective immunity. E-specific double-positive IFN-γ and TNF-α T-cells were induced in BALB/c mice after immunizations with a two-dose strategy. With the success of mRNA vaccine technology in facing the coronavirus (COVID-19) pandemic, our data support the development of prM-E RNActive ® as a promising mRNA vaccine against Zika to counter future epidemics.

Many different vaccine candidates against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, are currently approved and under development. Vaccine platforms vary from mRNA vaccines to viral-vectored vaccines, and several candidates have been shown to produce humoral and cellular responses in small animal models, non-human primates, and human volunteers. In this study, six non-human primates received a prime-boost intramuscular vaccination with 4 µg of mRNA vaccine candidate CV07050101, which encodes a pre-fusion stabilized spike (S) protein of SARS-CoV-2. Boost vaccination was performed 28 days post prime vaccination. As a control, six animals were similarly injected with PBS. Humoral and cellular immune responses were investigated at time of vaccination, and two weeks afterwards. No antibodies could be detected at two and four weeks after prime vaccination. Two weeks after boost vaccination, binding but no neutralizing antibodies were detected in four out of six non-human primates. SARS-CoV-2 S protein-specific T cell responses were detected in these four animals. In conclusion, prime-boost vaccination with 4 µg of vaccine candidate CV07050101 resulted in limited immune responses in four out of six non-human primates.

The ongoing SARS-CoV-2 pandemic necessitates the fast development of vaccines. Recently, viral mutants termed variants of concern (VOC) which may escape host immunity have emerged. The efficacy of spike encoding mRNA vaccines (CVnCoV and CV2CoV) against the ancestral strain and the VOC B.1.351 was tested in a K18-hACE2 transgenic mouse model. Naive mice and mice immunized with a formalin-inactivated SARS-CoV-2 preparation were used as controls. mRNA-immunized mice develop elevated SARS-CoV-2 RBD-specific antibody and neutralization titers which are readily detectable, but significantly reduced against VOC B.1.351. The mRNA vaccines fully protect from disease and mortality caused by either viral strain. SARS-CoV-2 remains undetected in swabs, lung, or brain in these groups. Despite lower neutralizing antibody titers compared to the ancestral strain BavPat1, CVnCoV and CV2CoV show complete disease protection against the novel VOC B.1.351 in our studies.

mRNA technologies have recently proven clinical efficacy against coronavirus disease 2019 and are among the most promising technologies to address the current pandemic. Here, we show preclinical data for our clinical candidate CVnCoV, a lipid nanoparticle-encapsulated mRNA vaccine that encodes full-length, pre-fusion stabilised severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein. In contrast to previously published approaches, CVnCoV is exclusively composed of naturally occurring nucleotides. Immunisation with CVnCoV induced strong humoral responses with high titres of virus-neutralising antibodies and robust T-cell responses. CVnCoV vaccination protected hamsters from challenge with wild-type SARS-CoV-2, demonstrated by the absence of viral replication in the lungs. Hamsters vaccinated with a suboptimal dose of CVnCoV leading to breakthrough viral replication exhibited no evidence of vaccine-enhanced disease. Overall, data presented here provide evidence that CVnCoV represents a potent and safe vaccine candidate against SARS-CoV-2.

: There is a global need for effective and affordable rabies vaccines, which is unmet by current vaccines due to limitations in their production capacities, required administration schedules, storage requirements, and cost. Many different experimental approaches previously used for bacterial and viral vaccines have been applied to rabies, but with variable success. One of the most promising new concepts is the use of messenger RNA (mRNA) in encoding the main rabies virus antigen, the envelope glycoprotein (RABV-G). CureVac has applied their proprietary technology platform for the production of mRNA to this problem, resulting in the rabies vaccine candidate CV7201. Following preclinical studies in mice and pigs showing that CV7201 could induce neutralizing immune responses that protected against rabies virus, different dosages and routes of administration of CV7201 were tested in a phase 1 human study. This clinical study proved that mRNA vaccination was safe and had an acceptable reactogenicity profile, but immune responses depended on the mode of administration, and they did not unequivocally support CV7201 for further development as a prophylactic vaccine with this particular formulation. Further, preclinical studies using RABV-G mRNA encapsulated in lipid nanoparticles (LNPs) showed an improved response in both mice and nonhuman primates, and these encouraging results are currently being followed up in clinical studies in humans. This review summarizes the recent advances in mRNA vaccines against rabies., Competing Interests: Authors are employees of CureVac AG, a company developing mRNA-based vaccines and therapeutics. E.D. and B.P. are inventors on several patent applications on mRNA-based vaccines.

First attempts to use exogenous mRNA for protein expression in vivo were made more than 25 years ago. However, widespread appreciation of in vitro transcribed mRNA as a powerful technology for supplying therapeutic proteins to the body has evolved only during the past few years. Various approaches to turning mRNA into a potent therapeutic have been developed. All of them share utilization of specifically designed, rather than endogenous, sequences and thorough purification protocols. Apart from this, there are two fundamental philosophies, one promoting the use of chemically modified nucleotides, the other advocating restriction to unmodified building blocks. Meanwhile, both strategies have received broad support by successful mRNA-based protein treatments in animal models. For such in vivo use, specifically optimized mRNA had to be combined with potent formulations to enable efficient in vivo delivery. The present review analyzes the applicability of mRNA technology to antibody therapy in all main fields: antitoxins, infectious diseases, and oncology., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Ever since the development of the first vaccine more than 200 years ago, vaccinations have greatly decreased the burden of infectious diseases worldwide, famously leading to the eradication of small pox and allowing the restriction of diseases such as polio, tetanus, diphtheria, and measles. A multitude of research efforts focuses on the improvement of established and the discovery of new vaccines such as the HPV (human papilloma virus) vaccine in 2006. However, radical changes in the density, age distribution and traveling habits of the population worldwide as well as the changing climate favor the emergence of old and new pathogens that bear the risk of becoming pandemic threats. In recent years, the rapid spread of severe infections such as HIV, SARS, Ebola, and Zika have highlighted the dire need for global preparedness for pandemics, which necessitates the extremely rapid development and comprehensive distribution of vaccines against potentially previously unknown pathogens. What is more, the emergence of antibiotic resistant bacteria calls for new approaches to prevent infections. Given these changes, established methods for the identification of new vaccine candidates are no longer sufficient to ensure global protection. Hence, new vaccine technologies able to achieve rapid development as well as large scale production are of pivotal importance. This review will discuss viral vector and nucleic acid-based vaccines (DNA and mRNA vaccines) as new approaches that might be able to tackle these challenges to global health.

Although effective rabies virus vaccines have been existing for decades, each year, rabies virus infections still cause around 50.000 fatalities worldwide. Most of these cases occur in developing countries, where these vaccines are not available. The reasons for this are the prohibitive high costs of cell culture or egg grown rabies virus vaccines and the lack of a functional cold chain in many regions in which rabies virus is endemic. Here, we describe the excellent temperature resistance of a non-replicating mRNA based rabies virus vaccine encoding the rabies virus glycoprotein (RABV-G). Prolonged storage of the vaccine from -80°C to up to +70°C for several months did not impact the protective capacity of the mRNA vaccine. Efficacy after storage was demonstrated by the induction of rabies specific virus neutralizing antibodies and protection in mice against lethal rabies infection. Moreover, storing the vaccine at oscillating temperatures between +4° and +56°C for 20 cycles in order to simulate interruptions of the cold chain during vaccine transport, did not affect the vaccine's immunogenicity and protective characteristics, indicating that maintenance of a cold chain is not essential for this vaccine.

Background: Prophylactic and therapeutic vaccines often depend upon a strong activation of the innate immune system to drive a potent adaptive immune response, often mediated by a strong adjuvant. For a number of adjuvants immunological readouts may not be consistent across species., Methods: In this study, we evaluated the innate immunostimulatory potential of mRNA vaccines in both humans and mice, using a novel mRNA-based vaccine encoding influenza A hemagglutinin of the pandemic strain H1N1pdm09 as a model. This evaluation was performed using an in vitro model of human innate immunity and in vivo in mice after intradermal injection., Results: Results suggest that immunostimulation from the mRNA vaccine in humans is similar to that in mice and acts through cellular RNA sensors, with genes for RLRs [ddx58 (RIG-1) and ifih1 (MDA-5)], TLRs (tlr3, tlr7, and tlr8-human only), and CLRs (clec4gp1, clec2d, cledl1) all significantly up-regulated by the mRNA vaccine. The up-regulation of TLR8 and TLR7 points to the involvement of both mDCs and pDCs in the response to the mRNA vaccine in humans. In both humans and mice activation of these pathways drove maturation and activation of immune cells as well as production of cytokines and chemokines known to attract and activate key players of the innate and adaptive immune system., Conclusion: This translational approach not only allowed for identification of the basic mechanisms of self-adjuvantation from the mRNA vaccine but also for comparison of the response across species, a response that appears relatively conserved or at least convergent between the in vitro human and in vivo mouse models.

Rabies is a zoonotic infectious disease of the central nervous system (CNS). In unvaccinated or untreated subjects, rabies virus infection causes severe neurological symptoms and is invariably fatal. Despite the long-standing existence of effective vaccines, vaccine availability remains insufficient, with high numbers of fatal infections mostly in developing countries. Nucleic acid based vaccines have proven convincingly as a new technology for the fast development of vaccines against newly emerging pathogens, diseases where no vaccine exists or for replacing already existing vaccines. We used an optimized non-replicating rabies virus glycoprotein (RABV-G) encoding messenger RNA (mRNA) to induce potent neutralizing antibodies (VN titers) in mice and domestic pigs. Functional antibody titers were followed in mice for up to one year and titers remained stable for the entire observation period in all dose groups. T cell analysis revealed the induction of both, specific CD4+ as well as CD8+ T cells by RABV-G mRNA, with the induced CD4+ T cells being higher than those induced by a licensed vaccine. Notably, RABV-G mRNA vaccinated mice were protected against lethal intracerebral challenge infection. Inhibition of viral replication by vaccination was verified by qRT-PCR. Furthermore, we demonstrate that CD4+ T cells are crucial for the generation of neutralizing antibodies. In domestic pigs we were able to induce VN titers that correlate with protection in adult and newborn pigs. This study demonstrates the feasibility of a non-replicating mRNA rabies vaccine in small and large animals and highlights the promises of mRNA vaccines for the prevention of infectious diseases.

Nucleotide based vaccines represent an enticing, novel approach to vaccination. We have developed a novel immunization technology, RNActive(®) vaccines, that have two important characteristics: mRNA molecules are used whose protein expression capacity has been enhanced by 4 to 5 orders of magnitude by modifications of the nucleotide sequence with the naturally occurring nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine) that do not affect the primary amino acid sequence. Second, they are complexed with protamine and thus activate the immune system by involvement of toll-like receptor (TLR) 7. Essentially, this bestows self-adjuvant activity on RNActive(®) vaccines. RNActive(®) vaccines induce strong, balanced immune responses comprising humoral and cellular responses, effector and memory responses as well as activation of important subpopulations of immune cells, such as Th1 and Th2 cells. Pre-germinal center and germinal center B cells were detected in human patients upon vaccination. RNActive(®) vaccines successfully protect against lethal challenges with a variety of different influenza strains in preclinical models. Anti-tumor activity was observed preclinically under therapeutic as well as prophylactic conditions. Initial clinical experiences suggest that the preclinical immunogenicity of RNActive(®) could be successfully translated to humans.

Despite substantial improvements, influenza vaccine production-and availability-remain suboptimal. Influenza vaccines based on mRNA may offer a solution as sequence-matched, clinical-grade material could be produced reliably and rapidly in a scalable process, allowing quick response to the emergence of pandemic strains. Here we show that mRNA vaccines induce balanced, long-lived and protective immunity to influenza A virus infections in even very young and very old mice and that the vaccine remains protective upon thermal stress. This vaccine format elicits B and T cell-dependent protection and targets multiple antigens, including the highly conserved viral nucleoprotein, indicating its usefulness as a cross-protective vaccine. In ferrets and pigs, mRNA vaccines induce immunological correlates of protection and protective effects similar to those of a licensed influenza vaccine in pigs. Thus, mRNA vaccines could address substantial medical need in the area of influenza prophylaxis and the broader realm of anti-infective vaccinology.

The blood-brain barrier (BBB) facilitates amyloid-β (Aβ) exchange between the blood and the brain. Here, we found that the cellular prion protein (PrP(c)), a putative receptor implicated in mediating Aβ neurotoxicity in Alzheimer's disease (AD), participates in Aβ transcytosis across the BBB. Using an in vitro BBB model, [(125)I]-Aβ(1-40) transcytosis was reduced by genetic knockout of PrP(c) or after addition of a competing PrP(c)-specific antibody. Furthermore, we provide evidence that PrP(c) is expressed in endothelial cells and, that monomeric Aβ(1-40) binds to PrP(c). These observations provide new mechanistic insights into the role of PrP(c) in AD.

Elucidation of the structure of PrP(Sc) continues to be one major challenge in prion research. The mechanism of propagation of these infectious agents will not be understood until their structure is solved. Given that high resolution techniques such as NMR or X-ray crystallography cannot be used, a number of lower resolution analytical approaches have been attempted. Thus, limited proteolysis has been successfully used to pinpoint flexible regions within prion multimers (PrP(Sc)). However, the presence of covalently attached sugar antennae and glycosylphosphatidylinositol (GPI) moieties makes mass spectrometry-based analysis impractical. In order to surmount these difficulties we analyzed PrP(Sc) from transgenic mice expressing prion protein (PrP) lacking the GPI membrane anchor. Such animals produce prions that are devoid of the GPI anchor and sugar antennae, and, thereby, permit the detection and location of flexible, proteinase K (PK) susceptible regions by Western blot and mass spectrometry-based analysis. GPI-less PrP(Sc) samples were digested with PK. PK-resistant peptides were identified, and found to correspond to molecules cleaved at positions 81, 85, 89, 116, 118, 133, 134, 141, 152, 153, 162, 169 and 179. The first 10 peptides (to position 153), match very well with PK cleavage sites we previously identified in wild type PrP(Sc). These results reinforce the hypothesis that the structure of PrP(Sc) consists of a series of highly PK-resistant β-sheet strands connected by short flexible PK-sensitive loops and turns. A sizeable C-terminal stretch of PrP(Sc) is highly resistant to PK and therefore perhaps also contains β-sheet secondary structure.

The prion agent is the infectious particle causing spongiform encephalopathies in animals and humans and is thought to consist of an altered conformation (PrP(Sc)) of the normal and ubiquitous prion protein PrP(C). The interaction of the prion agent with the immune system, particularly the humoral immune response, has remained unresolved. Here we investigated the immunogenicity of full-length native and infectious prions, as well as the specific biological effects of the resulting monoclonal antibodies (MAbs) on the binding and clearance of prions in cell culture and in in vivo therapy. Immunization of prion knockout (Prnp(0/0)) mice with phosphotungstic acid-purified mouse prions resulted in PrP-specific monoclonal antibodies with binding specificities selective for PrP(Sc) or for both PrP(C) and PrP(Sc). PrP(Sc)-specific MAb W261, of the IgG1 isotype, reacted with prions from mice, sheep with scrapie, deer with chronic wasting disease (CWD), and humans with sporadic and variant Creutzfeldt-Jakob disease (CJD) in assays including a capture enzyme-linked immunosorbent assay (ELISA) system. This PrP(Sc)-specific antibody was unable to clear prions from mouse neuroblastoma cells (ScN2a) permanently infected with scrapie, whereas the high-affinity MAb W226, recognizing both isoforms, PrP(Sc) and PrP(C), did clear prions from ScN2a cells, as determined by a bioassay. However, an attempt to treat intraperitoneally prion infected mice with full-length W226 or with a recombinant variable-chain fragment (scFv) from W226 could only slightly delay the incubation time. We conclude that (i) native, full-length PrP(Sc) elicits a prion-specific antibody response in PrP knockout mice, (ii) a PrP(Sc)-specific antibody had no prion-clearing effect, and (iii) even a high-affinity MAb that clears prions in vitro (W226) may not necessarily protect against prion infection, contrary to previous reports using different antibodies.

Prions, the agents causing transmissible spongiform encephalopathies, colonize the brain of hosts after oral, parenteral, intralingual, or even transdermal uptake. However, prions are not generally considered to be airborne. Here we report that inbred and crossbred wild-type mice, as well as tga20 transgenic mice overexpressing PrP(C), efficiently develop scrapie upon exposure to aerosolized prions. NSE-PrP transgenic mice, which express PrP(C) selectively in neurons, were also susceptible to airborne prions. Aerogenic infection occurred also in mice lacking B- and T-lymphocytes, NK-cells, follicular dendritic cells or complement components. Brains of diseased mice contained PrP(Sc) and transmitted scrapie when inoculated into further mice. We conclude that aerogenic exposure to prions is very efficacious and can lead to direct invasion of neural pathways without an obligatory replicative phase in lymphoid organs. This previously unappreciated risk for airborne prion transmission may warrant re-thinking on prion biosafety guidelines in research and diagnostic laboratories.

The primary biological function of the endogenous cellular prion protein has remained unclear. We investigated its biological function in the generation of cellular immune responses using cellular prion protein gene-specific small interfering ribonucleic acid in vivo and in vitro. Our results were confirmed by blocking cellular prion protein with monovalent antibodies and by using cellular prion protein-deficient and -transgenic mice. In vivo prion protein gene-small interfering ribonucleic acid treatment effects were of limited duration, restricted to secondary lymphoid organs and resulted in a 70% reduction of cellular prion protein expression in leukocytes. Disruption of cellular prion protein signalling augmented antigen-specific activation and proliferation, and enhanced T cell receptor signalling, resulting in zeta-chain-associated protein-70 phosphorylation and nuclear factor of activated T cells/activator protein 1 transcriptional activity. In vivo prion protein gene-small interfering ribonucleic acid treatment promoted T cell differentiation towards pro-inflammatory phenotypes and increased survival of antigen-specific T cells. Cellular prion protein silencing with small interfering ribonucleic acid also resulted in the worsening of actively induced and adoptively transferred experimental autoimmune encephalomyelitis. Finally, treatment of myelin basic protein(1-11) T cell receptor transgenic mice with prion protein gene-small interfering ribonucleic acid resulted in spontaneous experimental autoimmune encephalomyelitis. Thus, central nervous system autoimmune disease was modulated at all stages of disease: the generation of the T cell effector response, the elicitation of T effector function and the perpetuation of cellular immune responses. Our findings indicate that cellular prion protein regulates T cell receptor-mediated T cell activation, differentiation and survival. Defects in autoimmunity are restricted to the immune system and not the central nervous system. Our data identify cellular prion protein as a regulator of cellular immunological homoeostasis and suggest cellular prion protein as a novel potential target for therapeutic immunomodulation.

The prion protein, PrP, exists in several stable conformations, with the presence of one conformation, PrP(Sc), associated with transmissible neurodegenerative diseases. Targeting PrP by high-affinity ligands has been proven to be an effective way of preventing peripheral prion infections. Here, we have generated bacterially expressed single chain fragments of the variable domains (scFv) of a monoclonal antibody in Escherichia coli, originally raised against purified PrP(Sc) that recognizes both PrP(C) and PrP(Sc). This scFv fragment had a dissociation constant (K(D)) with recombinant PrP of 2 nM and cleared prions in ScN2a cells at 4 nM, as demonstrated by a mouse prion bioassay. A peptide corresponding to the complementarity determining region 3 of the heavy chain (CDR3H) selectively bound PrP(Sc) but had lost antiprion activity. However, synthesis and application of an improved peptide mimicking side chain topology of CDR3H while exhibiting increased protease resistance, a retro-inverso d-peptide of CDR3H, still bound PrP(Sc) and reinstated antiprion activity. We conclude that (1) scFvW226 is so far the smallest polypeptide with bioassay confirmed antiprion activity, and (2) differential conformation specificity and bioactivity can be regulated by orchestrating the participation of different CDRs.

Three hundred seventy four patients with duodenal ulcers and Helicobacter pylori infections were given a four-week treatment of bismuth or ranitidin. In all patients two-week antibiotic therapy were given. Endoscopies with urease tests and histologic examinations were performed before initiation and four weeks after cessation of therapy. Four-week therapy with ranitidini and two-week therapy with amoxicillin and metronidasole is highly effective (89.6%) in duodenal ulcer healing and symptom improvement comparison to bismuth and antibiotic therapy.