A self-amplifying mRNA SARS-CoV-2 vaccine candidate induces safe and robust protective immunity in preclinical models.

RNA vaccines have demonstrated efficacy against SARS-CoV-2 in humans, and the technology is being leveraged for rapid emergency response. In this report, we assessed immunogenicity and, for the first time, toxicity, biodistribution, and protective efficacy in preclinical models of a two-dose self-amplifying messenger RNA (SAM) vaccine, encoding a prefusion-stabilized spike antigen of SARS-CoV-2 Wuhan-Hu-1 strain and delivered by lipid nanoparticles (LNPs). In mice, one immunization with the SAM vaccine elicited a robust spike-specific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched SARS-CoV-2 Wuhan strain as well as B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. High frequencies of spike-specific germinal center B, Th0/Th1 CD4, and CD8 T cell responses were observed in mice. Local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. In hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following SARS-CoV-2 challenge. Therefore, the SARS-CoV-2 SAM (LNP) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple SARS-CoV-2 variants, and has been advanced to phase 1 clinical evaluation (NCT04758962).

Repeated-Dose Toxicity, Biodistribution, and Shedding Assessments With a ChAd155 Respiratory Syncytial Virus Vaccine Candidate Evaluated in Rabbits and Rats.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections (LRTI) in infants, and toddlers and vaccines are not yet available. A pediatric RSV vaccine (ChAd155-RSV) is being developed to protect infants against RSV disease. The ChAd155-RSV vaccine consists of a recombinant replication-deficient chimpanzee-derived adenovirus (ChAd) group C vector engineered to express the RSV antigens F, N, and M2-1. The local and systemic effects of three bi-weekly intramuscular injections of the ChAd155-RSV vaccine was tested in a repeated-dose toxicity study in rabbits. After three intramuscular doses, the ChAd155-RSV vaccine was considered well-tolerated. Changes due to the vaccine-elicited inflammatory reaction/immune response were observed along with transient decreases in platelet count without physiological consequences, already reported for other adenovirus-based vaccines. In addition, the biodistribution and shedding of ChAd155-RSV were also characterized in two studies in rats. The distribution and persistence of the ChAd155-RSV vaccine candidate was consistent with other similar adenovector-based vaccines, with quantifiable levels of ChAd155-RSV observed at the injection site (muscle) and the draining lymph nodes up to 69 days post administration. The shedding results demonstrated that ChAd155-RSV was generally not detectable in any secretions or excreta samples. In conclusion, the ChAd155-RSV vaccine was well-tolerated locally and systemically.

Nonclinical safety assessment of repeated administration and biodistribution of ChAd3-EBO-Z Ebola candidate vaccine.

ChAd3-EBO-Z is an investigational adenovirus-based vaccine for the prevention of Ebola virus disease. Two nonclinical studies were performed to evaluate the biodistribution, local tolerance and potential local and systemic toxic effects of this vaccine. In the biodistribution study, rats received a single intramuscular injection of either ChAd3-EBO-Z or saline. Enlargement of the draining lymph nodes, starting on day 2, was noticed in ChAd3-EBO-Z-treated rats, indicating that an immune response had taken place. Viral DNA was mainly found at the injection sites and in the draining lymph nodes, from where it progressively disappeared during the observation period, while it was found only transiently and occasionally in other organs. In the repeated-dose toxicity study, either ChAd3-EBO-Z or saline was administered intramuscularly to rabbits on two occasions with a 2-week interval. General health status, rectal temperature, local tolerance, ophthalmology, hematology, coagulation and blood chemistry parameters were monitored. Macroscopic and microscopic evaluations were performed. Treatment-related changes included a transient increase in neutrophil count, C-reactive protein and fibrinogen levels, and a transient decrease in platelet count. As expected, microscopic observations 3 days after the second injection were related to the elicited inflammatory reaction, and these inflammatory responses had almost completely disappeared 29 days after the second immunization. In conclusion, the vaccine was locally and systemically well-tolerated and the viral vector was partially or totally cleared from the organs where it disseminated, supporting the clinical development of the vaccine.

Characterization of Pre-F-GCN4t, a Modified Human Respiratory Syncytial Virus Fusion Protein Stabilized in a Noncleaved Prefusion Conformation.

The human respiratory syncytial virus (hRSV) fusion (F) protein is considered a major target of the neutralizing antibody response to hRSV. This glycoprotein undergoes a major structural shift from the prefusion (pre-F) to the postfusion (post-F) state at the time of virus-host cell membrane fusion. Recent evidences suggest that the pre-F state is a superior target for neutralizing antibodies compared to the post-F state. Therefore, for vaccine purposes, we have designed and characterized a recombinant hRSV F protein, called Pre-F-GCN4t, stabilized in a pre-F conformation. To show that Pre-F-GCN4t does not switch to a post-F conformation, it was compared with a recombinant post-F molecule, called Post-F-XC. Pre-F-GCN4t was glycosylated and trimeric and displayed a conformational stability different from that of Post-F-XC, as shown by chemical denaturation. Electron microscopy analysis suggested that Pre-F-GCN4t adopts a lollipop-like structure. In contrast, Post-F-XC had a typical elongated conical shape. Hydrogen/deuterium exchange mass spectrometry demonstrated that the two molecules had common rigid folding core and dynamic regions and provided structural insight for their biophysical and biochemical properties and reactivity. Pre-F-GCN4t was shown to deplete hRSV-neutralizing antibodies from human serum more efficiently than Post-F-XC. Importantly, Pre-F-GCN4t was also shown to bind D25, a highly potent monoclonal antibody specific for the pre-F conformation. In conclusion, this construct presents several pre-F characteristics, does not switch to the post-F conformation, and presents antigenic features required for a protective neutralizing antibody response. Therefore, Pre-F-GCN4t can be considered a promising candidate vaccine antigen.IMPORTANCE Human respiratory syncytial virus (RSV) is a global leading cause of infant mortality and adult morbidity. The development of a safe and efficacious RSV vaccine remains an important goal. The RSV class I fusion (F) glycoprotein is considered one of the most promising vaccine candidates, and recent evidences suggest that the prefusion (pre-F) state is a superior target for neutralizing antibodies. Our study presents the physicochemical characterization of Pre-F-GCN4t, a molecule designed to be stabilized in the pre-F conformation. To confirm its pre-F conformation, Pre-F-GCN4t was analyzed in parallel with Post-F-XC, a molecule in the post-F conformation. Our results show that Pre-F-GCN4t presents characteristics of a stabilized pre-F conformation and support its use as an RSV vaccine antigen. Such an antigen may represent a significant advance in the development of an RSV vaccine.

Structural and computational design of a SARS-CoV-2 spike antigen with improved expression and immunogenicity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern challenge the efficacy of approved vaccines, emphasizing the need for updated spike antigens. Here, we use an evolutionary-based design aimed at boosting protein expression levels of S-2P and improving immunogenic outcomes in mice. Thirty-six prototype antigens were generated in silico and 15 were produced for biochemical analysis. S2D14, which contains 20 computationally designed mutations within the S2 domain and a rationally engineered D614G mutation in the SD2 domain, has an ~11-fold increase in protein yield and retains RBD antigenicity. Cryo-electron microscopy structures reveal a mixture of populations in various RBD conformational states. Vaccination of mice with adjuvanted S2D14 elicited higher cross-neutralizing antibody titers than adjuvanted S-2P against the SARS-CoV-2 Wuhan strain and four variants of concern. S2D14 may be a useful scaffold or tool for the design of future coronavirus vaccines, and the approaches used for the design of S2D14 may be broadly applicable to streamline vaccine discovery.

ChAd155-RSV vaccine is immunogenic and efficacious against bovine RSV infection-induced disease in young calves.

Respiratory syncytial virus (RSV) infection causes a substantial lower-respiratory-tract disease burden in infants, constituting a global priority for vaccine development. We evaluated immunogenicity, safety and efficacy of a chimpanzee adenovirus (ChAd)-based vaccine candidate, ChAd155-RSV, in a bovine RSV (bRSV) challenge model. This model closely reproduces the pathogenesis/clinical manifestations of severe pediatric RSV disease. In seronegative calves, ChAd155-RSV elicits robust neutralizing antibody responses against human RSV. Two doses protect calves from clinical symptoms/lung pathological changes, and reduce nasal/lung virus loads after both a short (4-week) and a long (16-week) interval between last immunization and subsequent bRSV challenge. The one-dose regimen confers near-complete or significant protection after short-term or long-term intervals before challenge, respectively. The presence of pre-existing bRSV-antibodies does not affect short-term efficacy of the two-dose regimen. Immunized calves present no clinical signs of enhanced respiratory disease. Collectively, this supports the development of ChAd155-RSV as an RSV vaccine candidate for infants.

Hamster neogenin, a host-cell protein contained in a respiratory syncytial virus candidate vaccine, induces antibody responses in rabbits but not in clinical trial participants.

A recombinant respiratory syncytial virus (RSV) fusion glycoprotein candidate vaccine (RSV-PreF) manufactured in Chinese hamster ovary cells was developed for immunization of pregnant women, to protect newborns against RSV disease through trans-placental antibody transfer. Traces of a host-cell protein, hamster neogenin (haNEO1), were identified in purified RSV-PreF antigen material. Given the high amino-acid sequence homology between haNEO1 and human neogenin (huNEO1), there was a risk that potential vaccine-induced anti-neogenin immunity could affect huNEO1 function in mother or fetus. Anti-huNEO1 IgGs were measured by enzyme-linked immunosorbent assay in sera from rabbits and trial participants (Phase 1 and 2 trials enrolling 128 men and 500 non-pregnant women, respectively; NCT01905215/NCT02360475) collected after immunization with RSV-PreF formulations containing different antigen doses with/without aluminum-hydroxide adjuvant. In rabbits, four injections administered at 14-day intervals induced huNEO1-specific IgG responses in an antigen-dose- and adjuvant-dependent manner, which plateaued in the highest-dose groups after three injections. In humans, no vaccination-induced anti-huNEO1 IgG responses were detected upon a single immunization, as the values in vaccine and control groups fluctuated around pre-vaccination levels up to 90/360 days post-vaccination. A minority of participants had anti-huNEO1 levels ≥ assay cutoff before vaccination, which did not increase post-vaccination. Thus, despite detecting vaccine-induced huNEO1-specific responses in rabbits, we found no evidence that the candidate vaccine had induced anti-huNEO1 immunity in human adults. The antigen purification process was nevertheless optimized, and haNEO1-reduced vaccines were used in a subsequent Phase 2 trial enrolling 400 non-pregnant women (NCT02956837), in which again no vaccine-induced anti-huNEO1 responses were detected.

Mek2 is dispensable for mouse growth and development.

MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase upon agonist binding to receptors. The ERK/MAP kinase cascade is involved in cell fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single Mek gene is present in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.

Pre-fusion RSV F strongly boosts pre-fusion specific neutralizing responses in cattle pre-exposed to bovine RSV.

Human respiratory syncytial virus (hRSV) is responsible for serious lower respiratory tract disease in infants and in older adults, and remains an important vaccine need. RSV fusion (F) glycoprotein is a key target for neutralizing antibodies. RSV F stabilized in its pre-fusion conformation (DS-Cav1 F) induces high neutralizing antibody titers in naïve animals, but it remains unknown to what extent pre-fusion F can boost pre-existing neutralizing responses in RSV seropositive adults. We here assess DS-Cav1 F immunogenicity in seropositive cattle pre-exposed to bovine RSV, a virus closely related to hRSV. A single immunization with non-adjuvanted DS-Cav1 F strongly boosts RSV neutralizing responses, directed towards pre-fusion F-specific epitopes, whereas a post-fusion F is unable to do so. Vaccination with pre-fusion F thus represents a promising strategy for maternal immunization and for other RSV vaccine target populations such as older adults.

A cell death pathway induced by antibody-mediated cross-linking of CD45 on lymphocytes.

The protein tyrosine phosphatase CD45 is a highly expressed glycoprotein present on all nucleated cells of hematopoietic origin. To date, all the functions attributed to CD45 are inherently coupled to its phosphatase activity. For instance, the regulation of lymphocyte antigen receptor signaling is mediated through the dephosphorylation, and hence activation, of Src-family kinases by CD45. Moreover, signaling via cytokine receptors is negatively modulated by CD45 by dephosphorylation of Janus kinase family members. Recently, another function for CD45, unrelated to regulation of surface receptor signaling, has been unraveled. Specific engagement of CD45 by monoclonal antibodies at the surface of lymphocytes induced their death, through an alternative caspase-independent pathway. In striking contrast to all other previously reported functions for CD45, its phosphatase activity is completely dispensable for the induction of cell death. This article reviews the current knowledge on the death pathway triggered by CD45 ligation on lymphocytes. In an attempt to better elucidate the mechanism of cell death induction through CD45, we also provide original data regarding the susceptibility of various subsets of immature and mature T and B cells to death induced by CD45 engagement. The physiological significance and therapeutic potential of CD45-induced death are also discussed.

High-throughput technology: green fluorescent protein to monitor cell death.

Reliable assessment of cell death is now pivotal to many research programs aiming at generating new antitumor compounds or at screening cDNA libraries to identify genes with pro- or antiapoptotic functions. Such approaches need to rely on reproducible, easy handling, and rapid microplate-based cytotoxicity assays that are amenable to high-throughput screening technologies. We describe here a method for the direct measurement of cell death, based on the detection of a decrease in fluorescence observed following death induction in cells stably expressing enhanced green fluorescent protein (EGFP). Our data clearly show that such a decrease in EGFP fluorescence after cell death induction happens in various cell types, including those routinely used in anticancer drug screening (i.e., murine and human, lymphoid, fibroblastic, or epithelial cell lines). Moreover, the decrease in EGFP fluorescence is observed in cells induced to die by a variety of apoptosis-inducing agents, such as glucocorticoids (dexamethasone), DNA- damaging agents (etoposide, cisplatin), microtubule disorganizers (paclitaxel), protein kinase C inhibitors (staurosporine), or a caspase-independent apoptotic stimulus (CD45 crosslinking). A decrease in fluorescence can be assessed either by flow cytometry or with a fluorescence microplate reader. The kinetics and specificity of this EGFP-based assay were comparable with those of other conventional techniques used to detect cell death. This novel EGFP-based microplate assay combines sensitivity and rapidity and is amenable to high-throughput setups, making it an assay of choice for evaluation of cell cytotoxicity.

Cellular specificity related to monoglyceride-induced cell death.

We have recently observed that monoglycerides (MGs), a family of lipids consisting of a single fatty acid moiety attached to a glycerol backbone, induce rapid dose-dependent apoptosis in murine thymocytes. In this work, we evaluated the sensitivity of various normal and malignant immune and non-immune cells to MGs. We demonstrate that the propensity to MG-induced death displayed by both T and B lymphocytes is clearly modulated according to their differentiation and activation status. For instance, the earliest T and B cell precursors are refractory to MG-mediated cell death. In the T-cell lineage, immature thymocytes are the most susceptible to MG treatment, while B cells from peripheral lymphoid organs appear more sensitive than B-cell subsets from the bone marrow. On the other hand, both activated T and B cells are more resistant to MG exposure than their non-activated counterparts. In addition, other hematopoietic lineages such as natural killer cells, macrophages, and erythroid cells are quite resistant to MG-induced death. Furthermore, using various immortalized cell lines from different tissues, we found that lymphomas and thymomas are the most sensitive among all lineages tested, while epithelial cells and fibroblasts are unaffected by MG treatment. Finally, MG-induced death was shown to be independent of Fas/Fas ligand (FasL) interactions. Altogether, our findings indicate that there is a cellular specificity related to MG-mediated cell death biased towards T and B lymphocytes. This suggests that MGs could potentially be used in the treatment of specific lymphoid disorders by bypassing the requirement for the Fas/FasL system.

An improved mouse model for endometriosis allows noninvasive assessment of lesion implantation and development.

OBJECTIVE: To test whether fragments of human endometrium transduced with the green fluorescent protein (GFP) cDNA and transplanted into nude mice can be noninvasively visualized. DESIGN: A murine experimental model for human endometriosis. SETTING: A biotechnology company. ANIMAL(S): Ovariectomized nude mice. INTERVENTION(S): Whole fragments of human endometrium were transduced in vitro by adenoviral infection with the GFP cDNA before transplantation into nude mice. Animals were noninvasively and repeatedly imaged before lesion collection. MAIN OUTCOME MEASURE(S): Fluorescence of GFP-expressing human endometrial fragments was evaluated before transplantation into animals. Development of endometriotic lesions was monitored through direct visualization of fluorescent tissue in the living animal or through conventional dissection. RESULT(S): GFP gene transfer into whole endometrial fragments can be performed, and a high proportion of cells express the reporter gene. Fluorescent endometrial fragments implant in nude mice and form endometriotic-like lesions, which can be directly visualized through the skin of living mice using a simple imaging device. CONCLUSION(S): This improved mouse model allows noninvasive and dynamic studies of lesion implantation and development to be conducted. In addition to helping better understand the pathophysiology of the disease, this model represents a valuable preclinical tool for testing the efficacy of new drugs targeting endometriosis, which should ultimately accelerate their development phase.

Monoglycerides induce apoptosis in human leukemic cells while sparing normal peripheral blood mononuclear cells.

A major drawback of the current antineoplastic treatments is their lack of specificity toward cancer cells, because they are most often cytotoxic to normal cells, thus creating related side effects. Hence, the identification of new apoptosis-inducing agents, specifically targeting malignant cells while sparing their normal counterparts, is of crucial interest. We show here that monoglycerides, a family of lipids consisting of a single fatty acid attached to a glycerol backbone, induce cell death in several human leukemic cell lines. Importantly, treatment of primary leukemic cells, obtained from B-cell chronic lymphocytic leukemia patients, resulted in rapid apoptosis. In striking contrast, resting or activated human peripheral blood mononuclear cells from healthy individuals were resistant to the same treatment. Therefore, these compounds could represent potential antileukemic drugs or could allow for the design of novel therapeutic agents applied to leukemia.

Serum concentrations of insulin-like growth factor-1, soluble tumor necrosis factor receptor-1 and angiogenin in endometriosis patients.

PROBLEM: Many soluble factors contributing to the pathophysiology of endometriosis are found at abnormal levels in patients suffering from the disease. We postulated that levels of these factors could also be altered in the serum of patients. We compared levels of insulin-like growth factor-1 (IGF-1), soluble form of tumor necrosis factor-alpha (TNF-alpha) receptor-1 (sTNFR-1) and angiogenin in the serum of patients with endometriosis and controls. METHOD OF STUDY: Levels of IGF-1, sTNFR-1 and angiogenin were measured by enzyme-linked immunosorbent assay in samples from 148 patients (77 cases and 71 controls) with diagnostic confirmed by laparoscopy. Correlations with demographic data and stage of the disease were evaluated and potential confounders in the study population were controlled. RESULTS: A significant increase in sTNFR-1 and angiogenin serum levels was observed in cases in comparison with controls, but only for patients in the follicular phase of the cycle. No significant difference was found in serum levels of IGF-1, sTNFR-1 and angiogenin between cases and controls in the luteal phase of the cycle. Correlations between levels of angiogenin and stage of the disease could also be observed. CONCLUSION: sTNFR-1 and angiogenin represent potential blood markers for endometriosis.

Apoptosis mediated through CD45 is independent of its phosphatase activity and association with leukocyte phosphatase-associated phosphoprotein.

Besides the well-recognized role of CD45 as a major player in TCR signaling, we and others have demonstrated that cross-linking of CD45 with mAbs can induce cell death in T lymphocytes. To investigate the role of CD45 phosphatase activity in apoptosis induction, we expressed either wild-type or phosphatase-dead CD45 molecules in a CD45-deficient BW5147 T cell line. We show here that the phosphatase activity of CD45 was not required for apoptosis triggering after cross-linking of the molecule. It is noteworthy that a revertant of the CD45-negative BW5147 cell line, expressing a truncated form of CD45 lacking most of the cytoplasmic domain, was also susceptible to CD45-mediated death. Moreover, we also demonstrate that leukocyte phosphatase-associated phosphoprotein expression is totally dispensable for CD45-mediated apoptosis to occur. Taken together, these results strongly suggest a role for the extracellular and/or the transmembrane portion of CD45 in apoptosis signaling, which contrasts with the previously reported functions for CD45 in T lymphocytes.

Quantitative assessment of human endometriotic tissue maintenance and regression in a noninvasive mouse model of endometriosis.

Endometriosis is a prevalent disease characterized by the estrogen-dependent ectopic growth of endometrial tissue. Most of the current medical therapies consist in inducing a hypoestrogenic state in patients, but these treatments are associated with severe side effects and high recurrence rates. The development of convenient and reliable endometriosis animal models would be instrumental to accelerate the emergence of new therapeutic alternatives. Recently, we developed an improved experimental model for endometriosis, relying on the infection of human endometrial fragments by an adenovirus carrying the green fluorescent protein. Following injection of fluorescent fragments into nude mice, the implantation and growth of endometriotic-like lesions could be followed noninvasively. In the present work, we demonstrate that this model can be used to quantify the size of fluorescent endometriotic lesions by in vivo imaging. To this end, we repeatedly measured lesion size over a 4-week period in mice supplemented or not with estradiol. The model was adequate to confirm previous results showing that estrogen is dispensable for the implantation phase of endometrial tissue, whereas it is required for lesion maintenance. As a proof of concept for inducing regression of established lesions, ganciclovir was used to treat animals implanted with human fluorescent endometrial fragments expressing thymidine kinase. A significant decrease in lesion size was observed by in vivo imaging in ganciclovir-treated mice. Together, the data indicate that the noninvasive animal model described here provides a tool for drug testing and/or gene target validation in endometriosis.