Intranasal vaccination with a lentiviral vector protects against SARS-CoV-2 in preclinical animal models.

To develop a vaccine candidate against coronavirus disease 2019 (COVID-19), we generated a lentiviral vector (LV) eliciting neutralizing antibodies against the Spike glycoprotein of SARS-CoV-2. Systemic vaccination by this vector in mice, in which the expression of the SARS-CoV-2 receptor hACE2 has been induced by transduction of respiratory tract cells by an adenoviral vector, confers only partial protection despite high levels of serum neutralizing activity. However, eliciting an immune response in the respiratory tract through an intranasal boost results in a >3 log10 decrease in the lung viral loads and reduces local inflammation. Moreover, both integrative and non-integrative LV platforms display strong vaccine efficacy and inhibit lung deleterious injury in golden hamsters, which are naturally permissive to SARS-CoV-2 replication and closely mirror human COVID-19 physiopathology. Our results provide evidence of marked prophylactic effects of LV-based vaccination against SARS-CoV-2 and designate intranasal immunization as a powerful approach against COVID-19.

Determinants Involved in Hepatitis C Virus and GB Virus B Primate Host Restriction.

UNLABELLED: Hepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts. IMPORTANCE: Hepatitis C virus (HCV) is an important human pathogen that infects over 150 million individuals worldwide and leads to chronic liver disease. The lack of a small animal model for this infection impedes the development of a preventive vaccine and pathogenesis studies. In seeking to establish a small primate model for HCV, we first attempted to generate recombinants between HCV and GB virus B (GBV-B), a hepacivirus that infects small New World primates (tamarins and marmosets). This approach revealed that the genetic distance between these hepaciviruses likely prevented virus morphogenesis. We next showed that HCV pseudoparticles were able to infect tamarin or marmoset hepatocytes efficiently, demonstrating that there was no restriction in HCV entry into these simian cells. Furthermore, we found that a highly cell culture-adapted HCV strain was able to achieve a complete viral cycle in primary marmoset hepatocyte cultures, providing a promising basis for further HCV adaptation to small primate hosts.

Kinetic characterization of PB1-F2-mediated immunopathology during highly pathogenic avian H5N1 influenza virus infection.

The PB1-F2 protein encoded by influenza A viruses can contribute to virulence, a feature that is dependent of its sequence polymorphism. Whereas PB1-F2 from some H1N1 viruses were shown to exacerbate the inflammatory response within the airways, the contribution of PB1-F2 to highly pathogenic avian influenza virus (HPAIV) virulence in mammals remains poorly described. Using a H5N1 HPAIV strain isolated from duck and its PB1-F2 knocked-out mutant, we characterized the dynamics of PB1-F2-associated host response in a murine model of lethal pneumonia. The mean time of death was 10 days for the two viruses, allowing us to perform global transcriptomic analyses and detailed histological investigations of the infected lungs at multiple time points. At day 2 post-infection (pi), while no histopathological lesion was observed, PB1-F2 expression resulted in a significant inhibition of cellular pathways involved in macrophage activation and in a transcriptomic signature suggesting that it promotes damage to the epithelial barrier. At day 4 pi, the gene profile associated with PB1-F2 expression revealed dysfunctions in NK cells activity. At day 8 pi, PB1-F2 expression was strongly associated with increased transcription of genes encoding chemokines and cytokines implicated in the recruitment of granulocytes, as well as expression of a number of genes encoding enzymes expressed by neutrophils. These transcriptomic data were fully supported by the histopathological analysis of the mice lungs which evidenced more severe inflammatory lesions and enhanced recruitment of neutrophils in the context of PB1-F2 expression, and thus provided a functional corroboration to the insight obtained in this work. In summary, our study shows that PB1-F2 of H5N1 HPAIV markedly influences the expression of the host transcriptome in a different way than its H1N1 counterparts: H5N1 PB1-F2 first delays the initial immune response but increases the pulmonary inflammatory response during the late stages of infection.

A human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes.

Human coronaviruses are associated with upper respiratory tract infections that occasionally spread to the lungs and other organs. Although airway epithelial cells represent an important target for infection, the respiratory epithelium is also composed of an elaborate network of dendritic cells (DCs) that are essential sentinels of the immune system, sensing pathogens and presenting foreign antigens to T lymphocytes. In this report, we show that in vitro infection by human coronavirus 229E (HCoV-229E) induces massive cytopathic effects in DCs, including the formation of large syncytia and cell death within only few hours. In contrast, monocytes are much more resistant to infection and cytopathic effects despite similar expression levels of CD13, the membrane receptor for HCoV-229E. While the differentiation of monocytes into DCs in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 requires 5 days, only 24 h are sufficient for these cytokines to sensitize monocytes to cell death and cytopathic effects when infected by HCoV-229E. Cell death induced by HCoV-229E is independent of TRAIL, FasL, tumor necrosis factor alpha, and caspase activity, indicating that viral replication is directly responsible for the observed cytopathic effects. The consequence of DC death at the early stage of HCoV-229E infection may have an impact on the early control of viral dissemination and on the establishment of long-lasting immune memory, since people can be reinfected multiple times by HCoV-229E.

Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a pH- and cysteine protease-independent FcγR pathway.

Public health measures successfully contained outbreaks of the severe acute respiratory syndrome coronavirus (SARS-CoV) infection. However, the precursor of the SARS-CoV remains in its natural bat reservoir, and reemergence of a human-adapted SARS-like coronavirus remains a plausible public health concern. Vaccination is a major strategy for containing resurgence of SARS in humans, and a number of vaccine candidates have been tested in experimental animal models. We previously reported that antibody elicited by a SARS-CoV vaccine candidate based on recombinant full-length Spike-protein trimers potentiated infection of human B cell lines despite eliciting in vivo a neutralizing and protective immune response in rodents. These observations prompted us to investigate the mechanisms underlying antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro. We demonstrate here that anti-Spike immune serum, while inhibiting viral entry in a permissive cell line, potentiated infection of immune cells by SARS-CoV Spike-pseudotyped lentiviral particles, as well as replication-competent SARS coronavirus. Antibody-mediated infection was dependent on Fcγ receptor II but did not use the endosomal/lysosomal pathway utilized by angiotensin I converting enzyme 2 (ACE2), the accepted receptor for SARS-CoV. This suggests that ADE of SARS-CoV utilizes a novel cell entry mechanism into immune cells. Different SARS vaccine candidates elicit sera that differ in their capacity to induce ADE in immune cells despite their comparable potency to neutralize infection in ACE2-bearing cells. Our results suggest a novel mechanism by which SARS-CoV can enter target cells and illustrate the potential pitfalls associated with immunization against it. These findings should prompt further investigations into SARS pathogenesis.

SUMOylation promotes PML degradation during encephalomyocarditis virus infection.

The promyelocytic leukemia (PML) protein is expressed in the diffuse nuclear fraction of the nucleoplasm and in matrix-associated structures, known as nuclear bodies (NBs). PML NB formation requires the covalent modification of PML to SUMO. The noncovalent interactions of SUMO with PML based on the identification of a SUMO-interacting motif within PML seem to be required for further recruitment within PML NBs of SUMOylated proteins. RNA viruses whose replication takes place in the cytoplasm and is inhibited by PML have developed various strategies to counteract the antiviral defense mediated by PML NBs. We show here that primary fibroblasts derived from PML knockout mice are more sensitive to infection with encephalomyocarditis virus (EMCV), suggesting that the absence of PML results in an increase in EMCV replication. Also, we found that EMCV induces a decrease in PML protein levels both in interferon-treated cells and in PMLIII-expressing cells. Reduction of PML was carried out by the EMCV 3C protease. Indeed, at early times postinfection, EMCV induced PML transfer from the nucleoplasm to the nuclear matrix and PML conjugation to SUMO-1, SUMO-2, and SUMO-3, leading to an increase in PML body size where the viral protease 3C and the proteasome component were found colocalizing with PML within the NBs. This process was followed by PML degradation occurring in a proteasome- and SUMO-dependent manner and did not involve the SUMO-interacting motif of PML. Together, these findings reveal a new mechanism evolved by EMCV to antagonize the PML pathway in the interferon-induced antiviral defense.

Prime and boost immunization with influenza and adenovirus encoding the Toxoplasma gondii surface antigen 2 (SAG2) induces strong protective immunity.

In this work, we explored an original vaccination protocol using recombinant influenza and adenovirus. We constructed recombinant influenza viruses harboring dicistronic NA segments containing the surface antigen 2 (SAG2) from Toxoplasma gondii under control of the duplicated 3' promoter. Recombinant influenza viruses were able to drive the expression of the foreign SAG2 sequence in cell culture and to replicate efficiently both in cell culture and in lungs of infected mice. In addition, mice primed with recombinant influenza virus and boosted with a recombinant adenovirus encoding SAG2 elicited both humoral and cellular immune responses specific for SAG2. Moreover, when immunized animals were challenged with the cystogenic P-Br strain of T. gondii, they displayed up to 85% of reduction in parasite burden. These results demonstrate the potential use of recombinant influenza vectors harboring the dicistronic segments in the development of vaccines against infectious diseases.

Plasmacytoid dendritic cells efficiently cross-prime naive T cells in vivo after TLR activation.

Cross-presentation is a crucial mechanism in tumoral and microbial immunity because it allows internalized cell associated or exogenous antigens (Ags) to be delivered into the major histocompatibility complex I pathway. This pathway is important for the development of CD8(+) T-cell responses and for the induction of tolerance. In mice, cross-presentation is considered to be a unique property of CD8alpha+ conventional dendritic cells (DCs). Here we show that splenic plasmacytoid DCs (pDCs) efficiently capture exogenous Ags in vivo but are not able to cross-present these Ags at steady state. However, in vitro and in vivo stimulation by Toll-like receptor-7, or -9 or viruses licenses pDCs to cross-present soluble or particulate Ags by a transporter associated with antigen processing-dependent mechanism. Induction of cross-presentation confers to pDCs the ability to generate efficient effector CD8+ T-cell responses against exogenous Ags in vivo, showing that pDCs may play a crucial role in induction of adaptive immune responses against pathogens that do not infect tissues of hemopoietic origin. This study provides the first evidence for an in vivo role of splenic pDCs in Ag cross-presentation and T-cell cross-priming and suggests that pDCs may constitute an attractive target to boost the efficacy of vaccines based on cytotoxic T lymphocyte induction.

Delivery of mengovirus-derived RNA replicons into tumoural liver enhances the anti-tumour efficacy of a peripheral peptide-based vaccine.

Hepatocellular carcinoma is a deadly cancer with growing incidence for which immunotherapy is one of the most promising therapeutic approach. Peptide-based vaccines designed to induce strong, sustained CD8+ T cell responses are effective in animal models and cancer patients. We demonstrated the efficacy of curative peptide-based immunisation against a unique epitope of SV40 tumour antigen, through the induction of a strong CD8+ T cell-specific response, in our liver tumour model. However, as in human clinical trials, most tumour antigen epitopes did not induce a therapeutic effect, despite inducing strong CD8+ T cell responses. We therefore modified the tumour environment to enhance peptide-based vaccine efficacy by delivering mengovirus (MV)-derived RNA autoreplicating sequences (MV-RNA replicons) into the liver. The injection of replication-competent RNA replicons into the liver converted partial tumour regression into tumour eradication, whereas non-replicating RNA had no such effect. Replicating RNA replicon injection induced local recruitment of innate immunity effectors (NK and NKT) to the tumour and did not affect specific CD8+ T cell populations or other myelolymphoid subsets. The local delivery of such RNA replicons into tumour stroma is therefore a promising strategy complementary to the use of peripheral peptide-based vaccines for treating liver tumours.

Antigen crosspresentation by human plasmacytoid dendritic cells.

Crosspresentation is a specialized function of myeloid dendritic cells (mDCs), allowing them to induce CD8+ T cell responses against exogenous antigens that are not directly produced in their cytotosol. Human plasmacytoid DCs (pDCs) are not considered so far as able to perform crosspresentation. We showed here that purified human pDCs crosspresented vaccinal lipopeptides and HIV-1 antigens from apoptotic cells to specific CD8+ T lymphocytes. Apoptotic debris were internalized by phagocytosis and the lipopeptide LPPol reached nonacidic endosomes. This crosspresentation was amplified upon influenza virus infection. Importantly, the efficiency of crosspresentation by pDCs was comparable to that of mDCs. This property of human pDCs needs to be taken into account to understand the pathogenesis of infectious, allergic, or autimmune diseases and to help achieve desired responses during vaccination by targeting specifically either type of DCs.

Heterologous viral RNA export elements improve expression of severe acute respiratory syndrome (SARS) coronavirus spike protein and protective efficacy of DNA vaccines against SARS.

The SARS-CoV spike glycoprotein (S) is the main target of the protective immune response in humans and animal models of SARS. Here, we demonstrated that efficient expression of S from the wild-type spike gene in cultured cells required the use of improved plasmid vectors containing donor and acceptor splice sites, as well as heterologous viral RNA export elements, such as the CTE of Mazon-Pfizer monkey virus or the PRE of Woodchuck hepatitis virus (WPRE). The presence of both splice sites and WPRE markedly improved the immunogenicity of S-based DNA vaccines against SARS. Upon immunization of mice with low doses (2 microg) of naked DNA, only intron and WPRE-containing vectors could induce neutralizing anti-S antibodies and provide protection against challenge with SARS-CoV. Our observations are likely to be useful for the construction of plasmid and viral vectors designed for optimal expression of intronless genes derived from cytoplasmic RNA viruses.

Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E.

Post-translational modifications and correct subcellular localization of viral structural proteins are prerequisites for assembly and budding of enveloped viruses. Coronaviruses, like the severe acute respiratory syndrome-associated virus (SARS-CoV), bud from the endoplasmic reticulum-Golgi intermediate compartment. In this study, the subcellular distribution and maturation of SARS-CoV surface proteins S, M and E were analysed by using C-terminally tagged proteins. As early as 30 min post-entry into the endoplasmic reticulum, high-mannosylated S assembles into trimers prior to acquisition of complex N-glycans in the Golgi. Like S, M acquires high-mannose N-glycans that are subsequently modified into complex N-glycans in the Golgi. The N-glycosylation profile and the absence of O-glycosylation on M protein relate SARS-CoV to the previously described group 1 and 3 coronaviruses. Immunofluorescence analysis shows that S is detected in several compartments along the secretory pathway from the endoplasmic reticulum to the plasma membrane while M predominantly localizes in the Golgi, where it accumulates, and in trafficking vesicles. The E protein is not glycosylated. Pulse-chase labelling and confocal microscopy in the presence of protein translation inhibitor cycloheximide revealed that the E protein has a short half-life of 30 min. E protein is found in bright perinuclear patches colocalizing with endoplasmic reticulum markers. In conclusion, SARS-CoV surface proteins S, M and E show differential subcellular localizations when expressed alone suggesting that additional cellular or viral factors might be required for coordinated trafficking to the virus assembly site in the endoplasmic reticulum-Golgi intermediate compartment.

Involvement of toll-like receptor 3 in the immune response of lung epithelial cells to double-stranded RNA and influenza A virus.

Influenza A is a highly contagious single-stranded RNA virus that infects both the upper and lower respiratory tracts of humans. The host innate immune Toll-like receptor (TLR) 3 was shown previously in cells of myeloid origin to recognize the viral replicative, intermediate double-stranded RNA (dsRNA). Thus, dsRNA may be critical for the outcome of the infection. Here we first compared the activation triggered by either influenza A virus or dsRNA in pulmonary epithelial cells. We established that TLR3 is constitutively expressed in human alveolar and bronchial epithelial cells, and we describe its intracellular localization. Expression of TLR3 was positively regulated by the influenza A virus and by dsRNA but not by other inflammatory mediators, including bacterial lipopolysaccharide, the cytokines tumor necrosis factor-alpha and interleukin (IL)-1beta, and the protein kinase C activator phorbol 12-myristate 13-acetate. We also demonstrated that TLR3 contributes directly to the immune response of respiratory epithelial cells to influenza A virus and dsRNA, and we propose a molecular mechanism by which these stimuli induce epithelial cell activation. This model involves mitogen-activated protein kinases, phosphatidylinositol 3-kinase/Akt signaling, and the TLR3-associated adaptor molecule TRIF but not MyD88-dependent activation of the transcription factors NF-kappaB or interferon regulatory factor/interferon-sensitive response-element pathways. Ultimately, this signal transduction elicits an epithelial response that includes the secretion of the cytokines IL-8, IL-6, RANTES (regulated on activation normal T cell expressed and secreted), and interferon-beta and the up-regulation of the major adhesion molecule ICAM-1.