Patients with IgE antibodies against the carbohydrate epitope galactose‐α‐1,3‐galactose (α‐Gal) have reported severe allergic reactions after consumption of red meat. Investigations have revealed ...associations between IgE to α‐Gal and tick bites. We provide the first direct evidence that α‐Gal is present within ticks thus potentially explaining the relationship between tick exposure and sensitization to α‐Gal, with development of red meat allergy as a secondary phenomena. Serum from Swedish patients with delayed severe reactions to red meat was included in the study. A dose‐dependent inhibition of IgE responses to α‐Gal by the tick Ixodes ricinus is demonstrated. Furthermore, using cryostat‐cut sections of I. ricinus, we show that both a monoclonal and a polyclonal antibody against α‐Gal stains the gastrointestinal tract of the tick. The same pattern is seen when staining with patient sera IgE positive to α‐Gal. These results confirm that the α‐Gal epitope is present in I. ricinus and imply host exposure to α‐Gal during a tick bite. This provides further evidence that tick bites are associated with IgE responses to α‐Gal and red meat allergy.
Crimean-Congo hemorrhagic fever (CCHF), caused by Crimean-Congo hemorrhagic fever virus (CCHFV), is on the World Health Organizations' list of prioritized diseases and pathogens. With global ...distribution, high fatality rate, and no approved vaccine or effective treatment, CCHF constitutes a threat against global health. In the current study, we demonstrate that vaccination with nucleoside-modified mRNA-lipid nanoparticles (mRNA-LNP), encoding for the CCHFV nucleoprotein (N) or glycoproteins (GcGn) protect IFNAR
mice against lethal CCHFV infection. In addition, we found that both mRNA-LNP induced strong humoral and cellular immune responses in IFNAR
and immunocompetent mice and that neutralizing antibodies are not necessary for protection. When evaluating immune responses induced by immunization including CCHFV Gc and Gn antigens, we found the Gc protein to be more immunogenic compared with the Gn protein. Hepatic injury is prevalent in CCHF and contributes to the severity and mortality of the disease in humans. Thus, to understand the immune response in the liver after infection and the potential effect of the vaccine, we performed a proteomic analysis on liver samples from vaccinated and control mice after CCHFV infection. Similar to observations in humans, vaccination affected the metabolic pathways. In conclusion, this study shows that a CCHFV mRNA-LNP vaccine, based on viral nucleo- or glycoproteins, mediate protection against CCHFV induced disease. Consequently, genetic immunization is an attractive approach to prevent disease caused by CCHFV and we believe we have necessary evidence to bring this vaccine platform to the next step in the development of a vaccine against CCHFV infection.
Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic pathogen causing Crimean-Congo hemorrhagic fever (CCHF), a severe fever disease. CCHFV has a wide distribution and is endemic in several areas around the world. Cases of CCHF are also being reported in new areas, indicating an expansion of the disease, which is of high concern. Dispersion of the disease, high fatality rate, and no approved vaccine makes CCHF a threat to global health. The development of a vaccine is thus of great importance. Here we show 100% protection against lethal CCHFV infection in mice immunized with mRNA-LNP encoding for different CCHFV proteins. The vaccination showed both robust humoral and cellular immunity. mRNA-LNP vaccines combine the ability to induce an effective immune response, the safety of a transient carrier, and the flexibility of genetic vaccines. This and our results from the current study support the development of a mRNA-LNP based vaccine against CCHFV.
Advanced therapy medicinal products (ATMP) are medicines for human use that are based on genes, cells or tissues. Over the past years, an increasing number of ATMP entered the market for treatment of ...cancer, genetic disorders, skeletal defects and metabolic diseases. However, the ATMP production methods often change from the initial concept to commercialization. This change is needed to improve the manufacturing feasibility for scaling up or scaling out. Moreover, the production must adhere to current good manufacturing practices (GMP), and needs to follow a risk-based approach, which often is challenging to implement due to the novelty of the products. Since most of the early ATMP development is done in academia, an environment that is not familiar with regulatory requirements for ATMP production in GMP, the initial manufacturing choice for pre-clinical studies is usually very different from what is required for clinical use. This leads to a lengthy production process optimization, unnecessary repetition of experiments and ultimately waste of funding. This consideration prompted us to provide an intermediate step between early ATMP production in research settings to GMP manufacturing. We built a dedicated facility, and we called this environment ‘pre-GMP’ to highlight that it is a step toward preparation to GMP manufacturing. This environment supports process development and provides a manufacturing fitness room before transferring to GMP suites. This paper addresses the relevance of pre-GMP, underlining the advantages and the possible disadvantages of this additional framework that may be key in accelerating the pace of ATMP toward clinic.
•Challenges faced during ATMP development in academia.•Experience in providing support to ATMP development by building and operating a pre-GMP core facility.•Consideration of the benefits of the pre-GMP model for other academic institutions.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing antibodies (NAbs) lack cross-reactivity between SARS-CoV species and variants and fail to mediate long-term ...protection against infection. The maintained protection against severe disease and death by vaccination suggests a role for cross-reactive T cells. We generated vaccines containing sequences from the spike or receptor binding domain, the membrane and/or nucleoprotein that induced only T cells, or T cells and NAbs, to understand their individual roles. In three models with homologous or heterologous challenge, high levels of vaccine-induced SARS-CoV-2 NAbs protected against neither infection nor mild histological disease but conferred rapid viral control limiting the histological damage. With no or low levels of NAbs, vaccine-primed T cells, in mice mainly CD8+ T cells, partially controlled viral replication and promoted NAb recall responses. T cells failed to protect against histological damage, presumably because of viral spread and subsequent T cell-mediated killing. Neither vaccine- nor infection-induced NAbs seem to provide long-lasting protective immunity against SARS-CoV-2. Thus, a more realistic approach for universal SARS-CoV-2 vaccines should be to aim for broadly cross-reactive NAbs in combination with long-lasting highly cross-reactive T cells. Long-lived cross-reactive T cells are likely key to prevent severe disease and fatalities during current and future pandemics.
Display omitted
Yan and colleagues explores the role of T cells to SARS-CoV-2 in three different challenge models. The study reveals that T cells alone have the ability to limit viral replication, but this may come at a cost of an increase tissue damage by cell killing.
New variants in the SARS‐CoV‐2 pandemic are more contagious (Alpha/Delta), evade neutralizing antibodies (Beta), or both (Omicron). This poses a challenge in vaccine development according to WHO. We ...designed a more universal SARS‐CoV‐2 DNA vaccine containing receptor‐binding domain loops from the huCoV‐19/WH01, the Alpha, and the Beta variants, combined with the membrane and nucleoproteins. The vaccine induced spike antibodies crossreactive between huCoV‐19/WH01, Beta, and Delta spike proteins that neutralized huCoV‐19/WH01, Beta, Delta, and Omicron virus in vitro. The vaccine primed nucleoprotein‐specific T cells, unlike spike‐specific T cells, recognized Bat‐CoV sequences. The vaccine protected mice carrying the human ACE2 receptor against lethal infection with the SARS‐CoV‐2 Beta variant. Interestingly, priming of cross‐reactive nucleoprotein‐specific T cells alone was 60% protective, verifying observations from humans that T cells protect against lethal disease. This SARS‐CoV vaccine induces a uniquely broad and functional immunity that adds to currently used vaccines.
Synopsis
This work describes the design and evaluation of a new type of genetic COVID‐19 vaccine, containing three binding domains from three SARS‐CoV‐2 variants combined with the M and the N proteins.
The DNA vaccine induces high levels of broadly cross‐reactive and neutralizing antibodies to multiple SARS‐CoV‐2 variants.
The vaccine primes broadly cross‐reactive T cells that even recognize Bat SARS‐COV sequences.
The vaccine completely protects K18 mice from lethal disease caused by a challenge with the SARS‐CoV‐2 Beta variant.
The data supports clinical data of this completely new DNA vaccine design.
This work describes the design and evaluation of a new type of genetic COVID‐19 vaccine, containing three binding domains from three SARS‐CoV‐2 variants combined with the M and the N proteins.
Hepatitis C virus (HCV) effectively establishes persistent infection in human livers. The non-structural (NS) 3/4A complex participates in this process by cleavage of interferon beta (IFN beta) ...promoter stimulator-1 (IPS-1; also termed Cardif/MAVS/VISA), which inhibits responses to double stranded (ds) RNA. However, it is not known whether this effect extends beyond innate responses.
To test if HCV NS3/4A affects innate and adaptive immune responses in vivo.
NS3 levels were semi-quantified in human liver biopsies, transfected cells, and in transgenic (Tg) mouse livers by western blot. The effect of NS3/4A on dsRNA-mediated signalling and on the integrity of IPS-1 was analysed using in vitro translation, transfected cells and Tg mice. Cytotoxic T cell (CTL)-mediated clearance of transient firefly luciferase (FLuc)- and/or NS3/4A-Tg hepatocytes was determined using in vivo imaging and western blot.
NS3 protein levels were in a comparable range (0.1-49 microg/g tissue) in infected human livers and Tg mouse livers. Importantly, these levels of NS3/4A reduced murine innate responses to synthetic dsRNA in vivo, supporting the possibility that this occurs also in infected humans. The likely explanation for this was the NS3/4A-mediated cleavage of mouse IPS-1, albeit less efficiently than human IPS-1. Despite this, FLuc- and/or NS3/4A-expressing murine hepatocytes were effectively eliminated by hepatic CTLs, utilising the classical molecules for virus-infected cell lysis, including CD8, IFN gamma, perforin and FasL.
Although HCV NS3/4A inhibits the innate immunity, this does not prevent CTL-mediated clearance of NS3/4A-expressing hepatocytes in vivo. Thus, other HCV proteins are most likely responsible for interfering with the adaptive immunity.
Background: The hepatitis C virus (HCV) mutates within human leucocyte antigen (HLA) class I restricted immunodominant epitopes of the non-structural (NS) 3/4A protease to escape cytotoxic T ...lymphocyte (CTL) recognition and promote viral persistence. However, variability is not unlimited, and sometimes almost absent, and factors that restrict viral variability have not been defined experimentally. Aims: We wished to explore whether the variability of the immunodominant CTL epitope at residues 1073–1081 of the NS3 protease was limited by viral fitness. Patients: Venous blood was obtained from six patients (four HLA-A2+) with chronic HCV infection and from one HLA-A2+ patient with acute HCV infection. Methods: NS3/4A genes were amplified from serum, cloned in a eukaryotic expression plasmid, sequenced, and expressed. CTL recognition of naturally occurring and artificially introduced escape mutations in HLA-A2-restricted NS3 epitopes were determined using CTLs from human blood and genetically immunised HLA-A2-transgenic mice. HCV replicons were used to test the effect of escape mutations on HCV protease activity and RNA replication. Results: Sequence analysis of NS3/4A confirmed low genetic variability. The major viral species had functional proteases with 1073–1081 epitopes that were generally recognised by cross reactive human and murine HLA-A2 restricted CTLs. Introduction of mutations at five positions of the 1073–1081 epitope prevented CTL recognition but three of these reduced protease activity and RNA replication. Conclusions: Viral fitness can indeed limit the variability of HCV within immunological epitopes. This helps to explain why certain immunological escape variants never appear as a major viral species in infected humans.
PDF
