MINCLE and TLR9 agonists synergize to induce Th1/Th17 vaccine memory and mucosal recall in mice and non-human primates.

Development of new vaccines tailored for difficult-to-target diseases is hampered by a lack of diverse adjuvants for human use, and none of the currently available adjuvants induce Th17 cells. Here, we develop a liposomal adjuvant, CAF®10b, that incorporates Mincle and Toll-like receptor 9 agonists. In parallel mouse and non-human primate studies comparing to CAF® adjuvants already in clinical trials, we report species-specific effects of adjuvant composition on the quality and magnitude of the responses. When combined with antigen, CAF®10b induces Th1 and Th17 responses and protection against a pulmonary infection with Mycobacterium tuberculosis in mice. In non-human primates, CAF®10b induces higher Th1 responses and robust Th17 responses detectable after six months, and systemic and pulmonary Th1 and Th17 recall responses, in a sterile model of local recall. Overall, CAF®10b drives robust memory antibody, Th1 and Th17 vaccine-responses via a non-mucosal immunization route across both rodent and primate species.

More rapid blood interferon α2 decline in fatal versus surviving COVID-19 patients.

Background: The clinical outcome of COVID-19 pneumonia is highly variable. Few biological predictive factors have been identified. Genetic and immunological studies suggest that type 1 interferons (IFN) are essential to control SARS-CoV-2 infection. Objective: To study the link between change in blood IFN-α2 level and plasma SARS-Cov2 viral load over time and subsequent death in patients with severe and critical COVID-19. Methods: One hundred and forty patients from the CORIMUNO-19 cohort hospitalized with severe or critical COVID-19 pneumonia, all requiring oxygen or ventilation, were prospectively studied. Blood IFN-α2 was evaluated using the Single Molecule Array technology. Anti-IFN-α2 auto-Abs were determined with a reporter luciferase activity. Plasma SARS-Cov2 viral load was measured using droplet digital PCR targeting the Nucleocapsid gene of the SARS-CoV-2 positive-strand RNA genome. Results: Although the percentage of plasmacytoid dendritic cells was low, the blood IFN-α2 level was higher in patients than in healthy controls and was correlated to SARS-CoV-2 plasma viral load at entry. Neutralizing anti-IFN-α2 auto-antibodies were detected in 5% of patients, associated with a lower baseline level of blood IFN-α2. A longitudinal analysis found that a more rapid decline of blood IFN-α2 was observed in fatal versus surviving patients: mortality HR=3.15 (95% CI 1.14-8.66) in rapid versus slow decliners. Likewise, a high level of plasma SARS-CoV-2 RNA was associated with death risk in patients with severe COVID-19. Conclusion: These findings could suggest an interest in evaluating type 1 IFN treatment in patients with severe COVID-19 and type 1 IFN decline, eventually combined with anti-inflammatory drugs. Clinical trial registration: https://clinicaltrials.gov, identifiers NCT04324073, NCT04331808, NCT04341584.

Early blood neurofilament light chain and myelin oligodendrocyte glycoprotein antibody levels associate with different disease courses of myelin oligodendrocyte glycoprotein-associated disease in children.

Acquired demyelinating syndrome associated with myelin oligodendrocyte glycoprotein antibodies, named recently myelin oligodendrocyte glycoprotein-associated disease, represents >27% of this paediatric syndrome. Relapses occur in 40% of them, which may be associated with severe outcomes. Aiming to identify biomarker allowing to predict relapse, we measured both myelin oligodendrocyte glycoprotein antibodies and neurofilament light chain levels in blood samples of patients that are known to reflect axonal injuries in neurological diseases including demyelinating autoimmune disorders. Three groups of patients were selected: relapsing myelin oligodendrocyte glycoprotein-associated disease (n = 8), non-relapsing myelin oligodendrocyte glycoprotein-associated disease (n = 7) and control patients with non-inflammatory neurological diseases (n = 12). Neurofilament light chain concentrations were measured in plasma of these three groups of patients using the high-sensitivity single-molecule array method at onset of the disease and 6 months later. At onset of the disease, we found that levels of neurofilament light chain in blood of non-relapsing patients were significantly higher than in control patients (means: 98.36 ± 22.66 versus 12.47 ± 2.47 pg/mL, **P < 0.01, Kruskal-Wallis test). The mean neurofilament light chain value in relapsing patients (82.16 ± 38.41 pg/mL) was not significantly different from that in non-relapsing and in control patients. Plasma myelin oligodendrocyte glycoprotein antibody levels were 2.5-fold higher in relapsing than in non-relapsing patients without reaching significance (means: 15.26 ± 4.87 versus 5.96 ± 1.13; two-tailed Mann-Whitney U-test P = 0.119). Plasma neurofilament light chain correlated significantly with myelin oligodendrocyte glycoprotein antibody levels in relapsing (two-tailed Spearman r = 0.8, P = 0.0218) but not in non-relapsing (two-tailed Spearman r = 0.17, P = 0.71). Interestingly, the ratio of neurofilament light chain-to-myelin oligodendrocyte glycoprotein antibodies was significantly lower in relapsing than in non-relapsing patients (means: 5.19 ± 1.61 versus 21.87 ± 6.13; two-tailed Mann-Whitney U-test P = 0.014). These findings suggest that measuring both neurofilament light chain and myelin oligodendrocyte glycoprotein antibody levels in patients at onset of demyelinating disease could predict relapse of myelin oligodendrocyte glycoprotein-associated disease.

A novel adjuvant formulation induces robust Th1/Th17 memory and mucosal recall responses in Non-Human Primates.

After clean drinking water, vaccination is the most impactful global health intervention. However, development of new vaccines against difficult-to-target diseases is hampered by the lack of diverse adjuvants for human use. Of particular interest, none of the currently available adjuvants induce Th17 cells. Here, we develop and test an improved liposomal adjuvant, termed CAF®10b, that incorporates a TLR-9 agonist. In a head-to-head study in non-human primates (NHPs), immunization with antigen adjuvanted with CAF®10b induced significantly increased antibody and cellular immune responses compared to previous CAF® adjuvants, already in clinical trials. This was not seen in the mouse model, demonstrating that adjuvant effects can be highly species specific. Importantly, intramuscular immunization of NHPs with CAF®10b induced robust Th17 responses that were observed in circulation half a year after vaccination. Furthermore, subsequent instillation of unadjuvanted antigen into the skin and lungs of these memory animals led to significant recall responses including transient local lung inflammation observed by Positron Emission Tomography-Computed Tomography (PET-CT), elevated antibody titers, and expanded systemic and local Th1 and Th17 responses, including >20% antigen-specific T cells in the bronchoalveolar lavage. Overall, CAF®10b demonstrated an adjuvant able to drive true memory antibody, Th1 and Th17 vaccine-responses across rodent and primate species, supporting its translational potential.

Rituximab Impairs B Cell Response But Not T Cell Response to COVID-19 Vaccine in Autoimmune Diseases.

OBJECTIVE: Antibody response to the messenger RNA (mRNA) COVID-19 vaccine has been shown to be diminished in rituximab (RTX)-treated patients. We undertook this study to compare humoral and T cell responses between healthy controls, patients with autoimmune diseases treated with RTX, and those treated with other immunosuppressants, all of whom had been vaccinated with 2 doses of the mRNA COVID-19 vaccine. METHODS: We performed anti-spike IgG and neutralization assays just before and 28 days after the second BNT162b2 (Pfizer-BioNTech) vaccine dose. The specific T cell response was assessed in activated CD4 and CD8 T cells using intracellular flow cytometry staining of cytokines (interferon-γ, tumor necrosis factor, and interleukin-2) after stimulation with SARS-CoV-2 spike peptide pools. RESULTS: A lower proportion of responders with neutralizing antibodies to the vaccine was observed in the RTX group (29%; n = 24) compared to the other immunosuppressants group (80%; n = 35) (P = 0.0001) and the healthy control group (92%; n = 26) (P < 0.0001). No patients treated with RTX in the last 6 months showed a response. Time since last infusion was the main factor influencing humoral response in RTX-treated patients. The functional CD4 and CD8 cellular responses to SARS-CoV-2 peptides for each single cytokine or polyfunctionality were not different in the RTX group compared to the other immunosuppressants group or the control group. In RTX-treated patients, the T cell response was not different between patients with and those without a humoral response. CONCLUSION: RTX induced a diminished antibody response to the mRNA COVID-19 vaccine, but the functional T cell response was not altered compared to healthy controls and autoimmune disease patients treated with other immunosuppressants. Further work is needed to assess the clinical protection granted by a functionally active T cell response in the absence of an anti-spike antibody response.

Response to COVID-19 mRNA vaccination in multiple myeloma is conserved but impaired compared to controls.

Patients with multiple myeloma are at high risk of severe forms of COVID-19. Despite data showing diminished response to vaccine, the era of highly efficient mRNA vaccine might be a gamechanger. We sought to examine response to mRNA vaccine between healthy controls (n = 28) and multiple myeloma (MM) patients (n = 27). Response was analyzed 1 month after the second dose of anti-SARS-CoV-2 BNT162b2 vaccine. Multiple myeloma patients showed diminished levels of Anti-Spike IgG levels compared to controls, but with a high proportion of patients achieving a humoral response (89% vs. 97% in controls). Neutralizing antibodies were present in 74% of patients versus 96% of controls. Patients under current daratumumab treatment had neutralizing activity of anti-SARS-CoV-2 antibodies. Multiple myeloma patients show diminished response to SARS-COV-2 vaccine but with still high response rate. The main potential risk factor of non-response to COVID-19 vaccine was uncontrolled disease under treatment.

Vaccine Inoculation Route Modulates Early Immunity and Consequently Antigen-Specific Immune Response.

Vaccination is one of the most efficient public healthcare measures to fight infectious diseases. Nevertheless, the immune mechanisms induced in vivo by vaccination are still unclear. The route of administration, an important vaccination parameter, can substantially modify the quality of the response. How the route of administration affects the generation and profile of immune responses is of major interest. Here, we aimed to extensively characterize the profiles of the innate and adaptive response to vaccination induced after intradermal, subcutaneous, or intramuscular administration with a modified vaccinia virus Ankara model vaccine in non-human primates. The adaptive response following subcutaneous immunization was clearly different from that following intradermal or intramuscular immunization. The subcutaneous route induced a higher level of neutralizing antibodies than the intradermal and intramuscular vaccination routes. In contrast, polyfunctional CD8+ T-cell responses were preferentially induced after intradermal or intramuscular injection. We observed the same dichotomy when analyzing the early molecular and cellular immune events, highlighting the recruitment of cell populations, such as CD8+ T lymphocytes and myeloid-derived suppressive cells, and the activation of key immunomodulatory gene pathways. These results demonstrate that the quality of the vaccine response induced by an attenuated vaccine is shaped by early and subtle modifications of the innate immune response. In this immunization context, the route of administration must be tailored to the desired type of protective immune response. This will be achieved through systems vaccinology and mathematical modeling, which will be critical for predicting the efficacy of the vaccination route for personalized medicine.

Optimize Prime/Boost Vaccine Strategies: Trained Immunity as a New Player in the Game.

Most vaccines require multiple doses to induce long-lasting protective immunity in a high frequency of vaccines, and to ensure strong both individual and herd immunity. Repetitive immunogenic stimulations not only increase the intensity and durability of adaptive immunity, but also influence its quality. Several vaccine parameters are known to influence adaptive immune responses, including notably the number of immunizations, the delay between them, and the delivery sequence of different recombinant vaccine vectors. Furthermore, the initial effector innate immune response is key to activate and modulate B and T cell responses. Optimization of homologous and heterologous prime/boost vaccination strategies requires a thorough understanding of how vaccination history affects memory B and T cell characteristics. This requires deeper knowledge of how innate cells respond to multiple vaccine encounters. Here, we review how innate cells, more particularly those of the myeloid lineage, sense and respond differently to a 1st and a 2nd vaccine dose, both in an extrinsic and intrinsic manner. On one hand, the presence of primary specific antibodies and memory T cells, whose critical properties change with time after priming, provides a distinct environment for innate cells at the time of re-vaccination. On the other hand, innate cells themselves can exert enhanced intrinsic antimicrobial functions, long after initial stimulation, which is referred to as trained immunity. We discuss the potential of trained innate cells to be game-changers in prime/boost vaccine strategies. Their increased functionality in antigen uptake, antigen presentation, migration, and as cytokine producers, could indeed improve the restimulation of primary memory B and T cells and their differentiation into potent secondary memory cells in response to the boost. A better understanding of trained immunity mechanisms will be highly valuable for harnessing the full potential of trained innate cells, to optimize immunization strategies.

The Route of Vaccine Administration Determines Whether Blood Neutrophils Undergo Long-Term Phenotypic Modifications.

Innate immunity modulates adaptive immunity and defines the magnitude, quality, and longevity of antigen-specific T- and B- cell immune memory. Various vaccine and administration factors influence the immune response to vaccination, including the route of vaccine delivery. We studied the dynamics of innate cell responses in blood using a preclinical model of non-human primates immunized with a live attenuated vaccinia virus, a recombinant Modified vaccinia virus Ankara (MVA) expressing a gag-pol-nef fusion of HIV-1, and mass cytometry. We previously showed that it induces a strong, early, and transient innate response, but also late phenotypic modifications of blood myeloid cells after two months when injected subcutaneously. Here, we show that the early innate effector cell responses and plasma inflammatory cytokine profiles differ between subcutaneous and intradermal vaccine injection. Additionally, we show that the intradermal administration fails to induce more highly activated/mature neutrophils long after immunization, in contrast to subcutaneous administration. Different batches of antibodies, staining protocols and generations of mass cytometers were used to generate the two datasets. Mass cytometry data were analyzed in parallel using the same analytical pipeline based on three successive clustering steps, including SPADE, and categorical heatmaps were compared using the Manhattan distance to measure the similarity between cell cluster phenotypes. Overall, we show that the vaccine per se is not sufficient for the late phenotypic modifications of innate myeloid cells, which are evocative of innate immune training. Its route of administration is also crucial, likely by influencing the early innate response, and systemic inflammation, and vaccine biodistribution.

Innate Molecular and Cellular Signature in the Skin Preceding Long-Lasting T Cell Responses after Electroporated DNA Vaccination.

DNA vaccines delivered with electroporation (EP) have shown promising results in preclinical models and are evaluated in clinical trials. In this study, we aim to characterize early mechanisms occurring in the skin after intradermal injection and EP of the auxoGTUmultiSIV DNA vaccine in nonhuman primates. First, we show that EP acts as an adjuvant by enhancing local inflammation, notably via granulocytes, monocytes/macrophages, and CD1aint-expressing cell recruitment. EP also induced Langerhans cell maturation, illustrated by CD86, CD83, and HLA-DR upregulation and their migration out of the epidermis. Second, we demonstrate the crucial role of the DNA vaccine in soluble factors release, such as MCP-1 or IL-15. Transcriptomic analysis showed that EP played a major role in gene expression changes postvaccination. However, the DNA vaccine is required to strongly upregulate several genes involved in inflammatory responses (e.g., Saa4), cell migration (e.g., Ccl3, Ccl5, or Cxcl10), APC activation (e.g., Cd86), and IFN-inducible genes (e.g., Ifit3, Ifit5, Irf7, Isg15, orMx1), illustrating an antiviral response signature. Also, AIM-2, a cytosolic DNA sensor, appeared to be strongly upregulated only in the presence of the DNA vaccine and trends to positively correlate with several IFN-inducible genes, suggesting the potential role of AIM-2 in vaccine sensing and the subsequent innate response activation leading to strong adaptive T cell responses. Overall, these results demonstrate that a combined stimulation of the immune response, in which EP and the auxoGTUmultiSIV vaccine triggered different components of the innate immunity, led to strong and persistent cellular recall responses.

Molecular and Cellular Dynamics in the Skin, the Lymph Nodes, and the Blood of the Immune Response to Intradermal Injection of Modified Vaccinia Ankara Vaccine.

New vaccine design approaches would be greatly facilitated by a better understanding of the early systemic changes, and those that occur at the site of injection, responsible for the installation of a durable and oriented protective response. We performed a detailed characterization of very early infection and host response events following the intradermal administration of the modified vaccinia virus Ankara as a live attenuated vaccine model in non-human primates. Integrated analysis of the data obtained from in vivo imaging, histology, flow cytometry, multiplex cytokine, and transcriptomic analysis using tools derived from systems biology, such as co-expression networks, showed a strong early local and systemic inflammatory response that peaked at 24 h, which was then progressively replaced by an adaptive response during the installation of the host response to the vaccine. Granulocytes, macrophages, and monocytoid cells were massively recruited during the local innate response in association with local productions of GM-CSF, IL-1ß, MIP1α, MIP1ß, and TNFα. We also observed a rapid and transient granulocyte recruitment and the release of IL-6 and IL-1RA, followed by a persistent phase involving inflammatory monocytes. This systemic inflammation was confirmed by molecular signatures, such as upregulations of IL-6 and TNF pathways and acute phase response signaling. Such comprehensive approaches improve our understanding of the spatiotemporal orchestration of vaccine-elicited immune response, in a live-attenuated vaccine model, and thus contribute to rational vaccine development.

The toxoplasma-host cell junction is anchored to the cell cortex to sustain parasite invasive force.

BACKGROUND: The public health threats imposed by toxoplasmosis worldwide and by malaria in sub-Saharan countries are directly associated with the capacity of their related causative agents Toxoplasma and Plasmodium, respectively, to colonize and expand inside host cells. Therefore, deciphering how these two Apicomplexan protozoan parasites access their host cells has been highlighted as a priority research with the perspective of designing anti-invasive molecules to prevent diseases. Central to the mechanism of invasion for both genera is mechanical force, which is thought to be applied by the parasite at the interface between the two cells following assembly of a unique cell-cell junction but this model lacks direct evidence and has been challenged by recent genetic studies. In this work, using parasites expressing the fluorescent core component of this junction, we analyze characteristic features of the kinematics of penetration of more than 1,000 invasion events. RESULTS: The majority of invasion events occur with a typical forward rotational progression of the parasite through a static junction into an invaginating host cell plasma membrane. However, if parasites encounter resistance and if the junction is not strongly anchored to the host cell cortex, as when parasites do not secrete the toxofilin protein and, therefore, are unable to locally remodel the cortical actin cytoskeleton, the junction travels retrogradely with the host cell membrane along the parasite surface allowing the formation of a functional vacuole. Kinetic measurements of the invasive trajectories strongly support a similar parasite driven force in both static and capped junctions, both of which lead to successful invasion. However, about 20% of toxofilin mutants fail to enter and eventually disengage from the host cell membrane while the secreted RhOptry Neck (RON2) molecules are posteriorally capped before being cleaved and released in the medium. By contrast in cells characterized by low cortex tension and high cortical actin dynamics junction capping and entry failure are drastically reduced. CONCLUSIONS: This kinematic analysis newly highlights that to invade cells parasites need to engage their motor with the junction molecular complex where force is efficiently applied only upon proper anchorage to the host cell membrane and cortex.