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Despite the high efficiency of current SARS-CoV-2 mRNA vaccines in reducing COVID-19 morbidity and mortality, waning immunity and the emergence of resistant variants underscore the need for novel vaccination strategies. This study explores a heterologous mRNA/Modified Vaccinia virus Ankara (MVA) prime/boost regimen employing a trimeric form of the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein compared to a homologous MVA/MVA regimen. In C57BL/6 mice, the RBD was delivered during priming via an mRNA vector encapsulated in nanoemulsions (NE) or lipid nanoparticles (LNP), followed by a booster with a replication-deficient MVA-based recombinant virus (MVA-RBD). This heterologous mRNA/MVA regimen elicited strong anti-RBD binding and neutralizing antibodies (BAbs and NAbs) against both the ancestral SARS-CoV-2 strain and different variants of concern (VoCs). Additionally, this protocol induced robust and polyfunctional RBD-specific CD4 and CD8 T cell responses, particularly in animals primed with mLNP-RBD. In K18-hACE2 transgenic mice, the LNP-RBD/MVA combination provided complete protection from morbidity and mortality following a live SARS-CoV-2 challenge compared with the partial protection observed with mNE-RBD/MVA or MVA/MVA regimens. Although the mNE-RBD/MVA regimen only protects half of the animals, it was able to induce antibodies with Fc-mediated effector functions besides NAbs. Moreover, viral replication and viral load in the respiratory tract were markedly reduced and decreased pro-inflammatory cytokine levels were observed. These results support the efficacy of heterologous mRNA/MVA vaccine combinations over homologous MVA/MVA regimen, using alternative nanocarriers that circumvent intellectual property restrictions of current mRNA vaccine formulations.

Abstract Vaccines based on mRNA technology have revolutionized the field. In fact, lipid nanoparticles (LNP) formulated with mRNA are the preferential vaccine platform used in the fight against SARS-CoV-2 infection, with wider application against other diseases. The high demand and property right protection of the most potent cationic/ionizable lipids used for LNP formulation of COVID-19 mRNA vaccines have promoted the design of alternative nanocarriers for nucleic acid delivery. In this study we have evaluated the immunogenicity and efficacy of different rationally designed lipid and polymeric-based nanoparticle prototypes against SARS-CoV-2 infection. An mRNA coding for a trimeric soluble form of the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 was encapsulated using different components to form nanoemulsions (NE), nanocapsules (NC) and lipid nanoparticles (LNP). The toxicity and biological activity of these prototypes were evaluated in cultured cells after transfection and in mice following homologous prime/boost immunization. Our findings reveal good levels of RBD protein expression with most of the formulations. In C57BL/6 mice immunized intramuscularly with two doses of formulated RBD-mRNA, the modified lipid nanoparticle (mLNP) and the classical lipid nanoparticle (LNP-1) were the most effective delivery nanocarriers at inducing binding and neutralizing antibodies against SARS-CoV-2. Both prototypes fully protected susceptible K18-hACE2 transgenic mice from morbidity and mortality following a SARS-CoV-2 challenge. These results highlight that modulation of mRNAs immunogenicity can be achieved by using alternative nanocarriers and support further assessment of mLNP and LNP-1 prototypes as delivery vehicles for mRNA vaccines.

Abstract Acute infection and chronic infection are the two most common fates of pathogenic virus infections. While several factors that contribute to these fates are described, the critical control points and the mechanisms that underlie infection fate regulation are incompletely understood. Using the acute and chronic lymphocytic choriomeningitis virus (LCMV) infection model of mice, we find that the early dynamic pattern of the IFN-I response is a differentiating trait between both infection fates. Acute-infected mice generate a 2-wave IFN-I response while chronic-infected mice generate only a 1-wave response. The underlying cause is a temporal difference in CD8 T cell-mediated killing of splenic marginal zone CD169+ macrophages. It occurs later in acute infection and thus enables CD169+ marginal zone macrophages to produce the 2nd IFN-I wave. This is required for subsequent immune events including induction of inflammatory macrophages, generation of effector CD8+ T cells and virus clearance. Importantly, these benefits come at a cost for the host in the form of spleen fibrosis. Due to an earlier marginal zone destruction, these ordered immune events are deregulated in chronic infection. Our findings demonstrate the critical importance of kinetically well-coordinated sequential immune events for acute infection control and highlights that it may come at a cost for the host organism.

Abstract Background Recombination is an important mechanism in the generation of genetic diversity of the human (HIV) and simian (SIV) immunodeficiency viruses. It requires the co-packaging of divergent RNA genomes into the same retroviral capsid and subsequent template switching during the reverse transcription reaction. By HIV-specific fluorescence in situ hybridization (FISH), we have previously shown that the splenocytes from 2 chronically infected patients with Castelman's disease were multi-infected and thus fulfill the in vivo requirements to generate genetic diversity by recombination. In order to analyze when multi-infection first occurs during a lentivirus infection and how the distribution of multi-infection evolves during the disease course, we now determined the SIV copy numbers from splenocytes of 11 SIVmac251-infected rhesus macaques cross-sectionally covering the time span of primary infection throughout to end-stage immunodeficiency. Results SIV multi-infection of single splenocytes was readily detected in all monkeys and all stages of the infection. Single-infected cells were more frequent than double- or triple- infected cells. There was no strong trend linking the copy number distribution to plasma viral load, disease stage, or CD4 cell counts. Conclusions SIV multi-infection of single cells is already established during the primary infection phase thus enabling recombination to affect viral evolution in vivo throughout the disease course.

Abstract Myxobacteria are amongst the top producers of natural products. The diversity and unique structural properties of their secondary metabolites is what make these social microbes highly attractive for drug discovery. Screening of products derived from these bacteria has revealed a puzzling amount of hits against infectious and non-infectious human diseases. Preying mainly on other bacteria and fungi, why would these ancient hunters manufacture compounds beneficial for us? The answer may be the targeting of shared processes and structural features conserved throughout evolution.

Abstract Background Investigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue. Methods Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay. Results IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6. Conclusion AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.

Abstract Latency is a major barrier towards virus elimination in HIV-1-infected individuals. Yet, the mechanisms that contribute to the maintenance of HIV-1 latency are incompletely understood. Here we describe the Schlafen 12 protein (SLFN12) as an HIV-1 restriction factor that establishes a post-transcriptional block in HIV-1-infected cells and thereby inhibits HIV-1 replication and virus reactivation from latently infected cells. The inhibitory activity is dependent on the HIV-1 codon usage and on the SLFN12 RNase active sites. Within HIV-1-infected individuals, SLFN12 expression in PBMCs correlated with HIV-1 plasma viral loads and proviral loads suggesting a link with the general activation of the immune system. Using an RNA FISH-Flow HIV-1 reactivation assay, we demonstrate that SLFN12 expression is enriched in infected cells positive for HIV-1 transcripts but negative for HIV-1 proteins. Thus, codon-usage dependent translation inhibition of HIV-1 proteins participates in HIV-1 latency and can restrict the amount of virus release after latency reversal.

Abstract Background The cellular proteins Pat1p, Lsm1p, and Dhh1p are required for the replication of some positive-strand viruses and therefore are potential targets for new antiviral drugs. To prioritize host targets for antiviral drug screening a comparative metabolome analysis in Saccharomyces cerevisiae reference strain BY4742 Matα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 and deletion strains pat1Δ, lsm1Δ and dhh1Δ was performed. Results GC/MS analysis permitted the quantification of 47 polar metabolites and the identification of 41 of them. Metabolites with significant variation between the strains were identified using partial least squares to latent structures discriminate analysis (PLS-DA). The analysis revealed least differences of pat1Δ to the reference strain as characterized by Euclidian distance of normalized peak areas. The growth rate and specific production rates of ethanol and glycerol were also most similar with this strain. Conclusion From these results we hypothesize that the human analog of yeast Pat1p is most likely the best drug target candidate.

Abstract The yeast Saccharomyces cerevisiae is a well-established model system for understanding fundamental cellular processes relevant to higher eukaryotic organisms. Less known is its value for virus research, an area in which Saccharomyces cerevisiae has proven to be very fruitful as well. The present review will discuss the main achievements of yeast-based studies in basic and applied virus research. These include the analysis of the function of individual proteins from important pathogenic viruses, the elucidation of key processes in viral replication through the development of systems that allow the replication of higher eukayotic viruses in yeast, and the use of yeast in antiviral drug development and vaccine production.

Abstract Background The human genome comprises numerous human endogenous retroviruses (HERVs) that formed millions of years ago in ancestral species. A number of loci of the HERV-K(HML-2) family are evolutionarily much younger. A recent study suggested an infectious HERV-K(HML-2) variant in humans and other primates. Isolating such a variant from human individuals would be a significant finding for human biology. Results When investigating expression patterns of specific HML-2 proviruses we encountered HERV-K(HML-2) cDNA sequences without proviral homologues in the human genome, named HERV-KX, that could very well support recently suggested infectious HML-2 variants. However, detailed sequence analysis, using the software RECCO, suggested that HERV-KX sequences were produced by recombination, possibly arising ex vivo, between transcripts from different HML-2 proviral loci. Conclusion As RT-PCR probably will be instrumental for isolating an infectious HERV-K(HML-2) variant, generation of "new" HERV-K(HML-2) sequences by ex vivo recombination seems inevitable. Further complicated by an unknown amount of allelic sequence variation in HERV-K(HML-2) proviruses, newly identified HERV-K(HML-2) variants should be interpreted very cautiously.

SARS-CoV-2 still presents a global threat to human health due to the continued emergence of new strains and waning immunity among vaccinated populations. Therefore, it is still relevant to investigate potential therapeutics, such as therapeutic interfering particles (TIPs). Mathematical and computational modeling are valuable tools to study viral infection dynamics for predictive analysis. Here, we expand on the previous work on SARS-CoV-2 intra-cellular replication dynamics to include defective interfering particles (DIPs) as potential therapeutic agents. We formulate a deterministic model that describes the replication of wild-type (WT) SARS-CoV-2 virus in the presence of DIPs. Sensitivity analysis of parameters to several model outputs is employed to inform us on those parameters to be carefully calibrated from experimental data. We then study the effects of co-infection on WT replication and how DIP dose perturbs the release of WT viral particles. Furthermore, we provide a stochastic formulation of the model that is compared to the deterministic one. These models could be further developed into population-level models or used to guide the development and dose of TIPs.

Immunotherapy with checkpoint inhibitors, albeit commonly used against tumors, is still at its infancy against chronic virus infections. It relies on the reinvigoration of exhausted T lymphocytes to eliminate virus-infected cells. Since T cell exhaustion is a physiological process to reduce immunopathology, the reinvigoration of these cells might be associated with an augmentation of pathological changes. To test this possibility, we here analyzed in the model system of chronic lymphocytic choriomeningitis virus (LCMV)-infected mice whether treatment with the checkpoint inhibitor anti-PD-L1 antibody would increase CD8 T cell-dependent fibrosis. We show that pre-existing spleen fibrosis did not worsen under conditions that increase CD8 T cell functionality and reduce virus loads suggesting that the CD8 T cell functionality increase remained below its pathogenicity threshold. These promising findings should further encourage immunotherapeutic trials against chronic virus infections.

Summary: SARS-CoV-2 emerged in December 2019 and quickly spread worldwide, continuously striking with an unpredictable evolution. Despite the success in vaccine production and mass vaccination programs, the situation is not still completely controlled, and therefore accessible second-generation vaccines are required to mitigate the pandemic. We previously developed an adjuvanted vaccine candidate coded PHH-1V, based on a heterodimer fusion protein comprising the RBD domain of two SARS-CoV-2 variants. Here, we report data on the efficacy, safety, and immunogenicity of PHH-1V in cynomolgus macaques. PHH-1V prime-boost vaccination induces high levels of RBD-specific IgG binding and neutralizing antibodies against several SARS-CoV-2 variants, as well as a balanced Th1/Th2 cellular immune response. Remarkably, PHH-1V vaccination prevents SARS-CoV-2 replication in the lower respiratory tract and significantly reduces viral load in the upper respiratory tract after an experimental infection. These results highlight the potential use of the PHH-1V vaccine in humans, currently undergoing Phase III clinical trials.

The COVID-19 pandemic has brought significant changes and advances in the field of vaccination, including the implementation and widespread use of encapsidated mRNA vaccines in general healthcare practice. Here, we present two new mRNAs expressing antigenic parts of the SARS-CoV-2 spike protein and provide data supporting their functionality. The first mRNA, called RBD-mRNA, encodes a trimeric form of the virus spike protein receptor binding domain (RBD). The other mRNA, termed T-mRNA, codes for the relevant HLA I and II spike epitopes. The two mRNAs (COVARNA mRNAs) were designed to be used for delivery to cells in combination, with the RBD-mRNA being the primary source of antigen and the T-mRNA working as an enhancer of immunogenicity by supporting CD4 and CD8 T-cell activation. This innovative approach substantially differs from other available mRNA vaccines, which are largely directed to antibody production by the entire spike protein. In this study, we first show that both mRNAs are functionally transfected into human antigen-presenting cells (APCs). We obtained peripheral blood mononuclear cell (PBMC) samples from three groups of voluntary donors differing in their immunity against SARS-CoV-2: non-infected (naïve), infected-recovered (convalescent), and vaccinated. Using an established method of co-culturing autologous human dendritic cells (hDCs) with T-cells, we detected proliferation and cytokine secretion, thus demonstrating the ability of the COVARNA mRNAs to activate T-cells in an antigen-specific way. Interestingly, important differences in the intensity of the response between the infected-recovered (convalescent) and vaccinated donors were observed, with the levels of T-cell proliferation and cytokine secretion (IFNγ, IL-2R, and IL-13) being higher in the vaccinated group. In summary, our data support the further study of these mRNAs as a combined approach for future use as a vaccine.

The lymphocytic choriomeningitis virus (LCMV) is a non-cytopathic virus broadly used in fundamental immunology as a mouse model for acute and chronic virus infections. LCMV remains a cause of meningitis in humans, in particular the fatal LCMV infection in organ transplant recipients, which highlights the pathogenic potential and clinical significance of this neglected human pathogen. Paradoxically, the kinetics of the LCMV intracellular life cycle has not been investigated in detail. In this study, we formulate and calibrate a mathematical model predicting the kinetics of biochemical processes, including the transcription, translation, and degradation of molecular components of LCMV underlying its replication in infected cells. The model is used to study the sensitivity of the virus growth, providing a clear ranking of intracellular virus replication processes with respect to their contribution to net viral production. The stochastic formulation of the model enables the quantification of the variability characteristics in viral production, probability of productive infection and secretion of protein-deficient viral particles. As it is recognized that antiviral therapeutic options in human LCMV infection are currently limited, our results suggest potential targets for antiviral therapies. The model provides a currently missing building module for developing multi-scale mathematical models of LCMV infection in mice.

Summary: Current COVID-19 vaccines have been associated with a decline in infection rates, prevention of severe disease, and a decrease in mortality rates. However, SARS-CoV-2 variants are continuously evolving, and development of new accessible COVID-19 vaccines is essential to mitigate the pandemic. Here, we present data on preclinical studies in mice of a receptor-binding domain (RBD)-based recombinant protein vaccine (PHH-1V) consisting of an RBD fusion heterodimer comprising the B.1.351 and B.1.1.7 SARS-CoV-2 variants formulated in SQBA adjuvant, an oil-in-water emulsion. A prime-boost immunisation with PHH-1V in BALB/c and K18-hACE2 mice induced a CD4+ and CD8+ T cell response and RBD-binding antibodies with neutralizing activity against several variants, and also showed a good tolerability profile. Significantly, RBD fusion heterodimer vaccination conferred 100% efficacy, preventing mortality in SARS-CoV-2 infected K18-hACE2 mice, but also reducing Beta, Delta and Omicron infection in lower respiratory airways. These findings demonstrate the feasibility of this recombinant vaccine strategy.

Summary: The contribution of cross-presenting XCR1+ dendritic cells (DCs) and SIRPα+ DCs in maintaining T cell function during exhaustion and immunotherapeutic interventions of chronic infections remains poorly characterized. Using the mouse model of chronic LCMV infection, we found that XCR1+ DCs are more resistant to infection and highly activated compared with SIRPα+ DCs. Exploiting XCR1+ DCs via Flt3L-mediated expansion or XCR1-targeted vaccination notably reinvigorates CD8+ T cells and improves virus control. Upon PD-L1 blockade, XCR1+ DCs are not required for the proliferative burst of progenitor exhausted CD8+ T (TPEX) cells but are indispensable to sustain the functionality of exhausted CD8+ T (TEX) cells. Combining anti-PD-L1 therapy with increased frequency of XCR1+ DCs improves functionality of TPEX and TEX subsets, while increase of SIRPα+ DCs dampened their proliferation. Together, this demonstrates that XCR1+ DCs are crucial for the success of checkpoint inhibitor-based therapies through differential activation of exhausted CD8+ T cell subsets.

The systemic bio-organization of humans and other mammals is essentially “preprogrammed”, and the basic interacting units, the cells, can be crudely mapped into discrete sets of developmental lineages and maturation states. Over several decades, however, and focusing on the immune system, we and others invoked evidence – now overwhelming – suggesting dynamic acquisition of cellular properties and functions, through tuning, re-networking, chromatin remodeling, and adaptive differentiation. The genetically encoded “algorithms” that govern the integration of signals and the computation of new states are not fully understood but are believed to be “smart”, designed to enable the cells and the system to discriminate meaningful perturbations from each other and from “noise”. Cellular sensory and response properties are shaped in part by recurring temporal patterns, or features, of the signaling environment. We compared this phenomenon to associative brain learning. We proposed that interactive cell learning is subject to selective pressures geared to performance, allowing the response of immune cells to injury or infection to be progressively coordinated with that of other cell types across tissues and organs. This in turn is comparable to supervised brain learning. Guided by feedback from both the tissue itself and the neural system, resident or recruited antigen-specific and innate immune cells can eradicate a pathogen while simultaneously sustaining functional homeostasis. As informative memories of immune responses are imprinted both systemically and within the targeted tissues, it is desirable to enhance tissue preparedness by incorporating attenuated-pathogen vaccines and informed choice of tissue-centered immunomodulators in vaccination schemes. Fortunately, much of the “training” that a living system requires to survive and function in the face of disturbances from outside or within is already incorporated into its design, so it does not need to deep-learn how to face a new challenge each time from scratch. Instead, the system learns from experience how to efficiently select a built-in strategy, or a combination of those, and can then use tuning to refine its organization and responses. Efforts to identify and therapeutically augment such strategies can take advantage of existing integrative modeling approaches. One recently explored strategy is boosting the flux of uninfected cells into and throughout an infected tissue to rinse and replace the infected cells.

A mathematical model of the human immunodeficiency virus Type 1 (HIV-1) life cycle in CD4 T cells was constructed and calibrated. It describes the activation of the intracellular Type I interferon (IFN-I) response and the IFN-induced suppression of viral replication. The model includes viral replication inhibition by interferon-induced antiviral factors and their inactivation by the viral proteins Vpu and Vif. Both deterministic and stochastic model formulations are presented. The stochastic model was used to predict efficiency of IFN-I-induced suppression of viral replication in different initial conditions for autocrine and paracrine effects. The probability of virion excretion for various MOIs and various amounts of IFN-I was evaluated and the statistical properties of the heterogeneity of HIV-1 and IFN-I production characterised.

SARS-CoV-2 still presents a global threat to human health due to the continued emergence of new strains and waning immunity among vaccinated populations. Therefore, it is still relevant to investigate potential therapeutics, such as therapeutic interfering particles (TIPs). Mathematical and computational modeling are valuable tools to study viral infection dynamics for predictive analysis. Here, we expand on the previous work on SARS-CoV-2 intra-cellular replication dynamics to include defective interfering particles (DIPs) as potential therapeutic agents. We formulate a deterministic model that describes the replication of wild-type (WT) SARS-CoV-2 virus in the presence of DIPs. Sensitivity analysis of parameters to several model outputs is employed to inform us on those parameters to be carefully calibrated from experimental data. We then study the effects of co-infection on WT replication and how DIP dose perturbs the release of WT viral particles. Furthermore, we provide a stochastic formulation of the model that is compared to the deterministic one. These models could be further developed into population-level models or used to guide the development and dose of TIPs. [ABSTRACT FROM AUTHOR]

Immunotherapy with checkpoint inhibitors, albeit commonly used against tumors, is still at its infancy against chronic virus infections. It relies on the reinvigoration of exhausted T lymphocytes to eliminate virus-infected cells. Since T cell exhaustion is a physiological process to reduce immunopathology, the reinvigoration of these cells might be associated with an augmentation of pathological changes. To test this possibility, we here analyzed in the model system of chronic lymphocytic choriomeningitis virus (LCMV)-infected mice whether treatment with the checkpoint inhibitor anti-PD-L1 antibody would increase CD8 T cell-dependent fibrosis. We show that pre-existing spleen fibrosis did not worsen under conditions that increase CD8 T cell functionality and reduce virus loads suggesting that the CD8 T cell functionality increase remained below its pathogenicity threshold. These promising findings should further encourage immunotherapeutic trials against chronic virus infections. [ABSTRACT FROM AUTHOR]

Mathematical modelling of infection processes in cells is of fundamental interest. It helps to understand the SARS-CoV-2 dynamics in detail and can be useful to define the vulnerability steps targeted by antiviral treatments. We previously developed a deterministic mathematical model of the SARS-CoV-2 life cycle in a single cell. Despite answering many questions, it certainly cannot accurately account for the stochastic nature of an infection process caused by natural fluctuation in reaction kinetics and the small abundance of participating components in a single cell. In the present work, this deterministic model is transformed into a stochastic one based on a Markov Chain Monte Carlo (MCMC) method. This model is employed to compute statistical characteristics of the SARS-CoV-2 life cycle including the probability for a non-degenerate infection process. Varying parameters of the model enables us to unveil the inhibitory effects of IFN and the effects of the ACE2 binding affinity. The simulation results show that the type I IFN response has a very strong effect on inhibition of the total viral progeny whereas the effect of a 10-fold variation of the binding rate to ACE2 turns out to be negligible for the probability of infection and viral production.