Design, immunogenicity, and efficacy of a pan-sarbecovirus dendritic-cell targeting vaccine.

BACKGROUND: There is an urgent need of a new generation of vaccine that are able to enhance protection against SARS-CoV-2 and related variants of concern (VOC) and emerging coronaviruses. METHODS: We identified conserved T- and B-cell epitopes from Spike (S) and Nucleocapsid (N) highly homologous to 38 sarbecoviruses, including SARS-CoV-2 VOCs, to design a protein subunit vaccine targeting antigens to Dendritic Cells (DC) via CD40 surface receptor (CD40.CoV2). FINDINGS: CD40.CoV2 immunization elicited high levels of cross-neutralizing antibodies against SARS-CoV-2, VOCs, and SARS-CoV-1 in K18-hACE2 transgenic mice, associated with viral control and survival after SARS-CoV-2 challenge. A direct comparison of CD40.CoV2 with the mRNA BNT162b2 vaccine showed that the two vaccines were equally immunogenic in mice. We demonstrated the potency of CD40.CoV2 to recall in vitro human multi-epitope, functional, and cytotoxic SARS-CoV-2 S- and N-specific T-cell responses that are unaffected by VOC mutations and cross-reactive with SARS-CoV-1 and, to a lesser extent, MERS epitopes. INTERPRETATION: We report the immunogenicity and antiviral efficacy of the CD40.CoV2 vaccine in a preclinical model providing a framework for a pan-sarbecovirus vaccine. FUNDINGS: This work was supported by INSERM and the Investissements d'Avenir program, Vaccine Research Institute (VRI), managed by the ANR and the CARE project funded from the Innovative Medicines Initiative 2 Joint Undertaking (JU).

TLR9- and CD40-Targeting Vaccination Promotes Human B Cell Maturation and IgG Induction via pDC-Dependent Mechanisms in Humanized Mice.

Mice reconstituted with a human immune system (humanized mice) provide a robust model to study human immunology, vaccinology, and human infectious diseases. However, the development and function of B cells in humanized mice is impaired. B cells from humanized mice are immature and are impaired in IgM to IgG isotype switch in response to infection or vaccination. In the present study we report that Toll-like receptor 9 (TLR9) agonist CpG-B combined with CD40-targeting vaccination triggered human B cell immunoglobin class-switch from IgM+ to IgG+ B cells in humanized mice. Human B cells from mice vaccinated with CpG-B as adjuvant were more mature in phenotype and produced significant levels of both total IgG and antigen-specific IgG. We found that CpG-B treatment activated human pDCs (plasmacytoid dendritic cells) in vivo to induce interferon-alpha (IFN-α)expression in humanized mice. Pre-depletion of human pDC in vivo abrogated the adjuvant effect of CpG-B. Our results indicate that TLR9 and CD40-targeting vaccination triggers human B cell maturation and immunoglobulin class-switch in a pDC-dependent manner in humanized mice. The findings also shed light on induction of human IgG antibodies in humanized mouse models.

TLR-9 agonist and CD40-targeting vaccination induces HIV-1 envelope-specific B cells with a diversified immunoglobulin repertoire in humanized mice.

The development of HIV-1 vaccines is challenged by the lack of relevant models to accurately induce human B- and T-cell responses in lymphoid organs. In humanized mice reconstituted with human hematopoietic stem cells (hu-mice), human B cell-development and function are impaired and cells fail to efficiently transition from IgM B cells to IgG B cells. Here, we found that CD40-targeted vaccination combined with CpG-B adjuvant overcomes the usual defect of human B-cell switch and maturation in hu-mice. We further dissected hu-B cell responses directed against the HIV-1 Env protein elicited by targeting Env gp140 clade C to the CD40 receptor of antigen-presenting cells. The anti-CD40.Env gp140 vaccine was injected with CpG-B in a homologous prime/boost regimen or as a boost of a NYVAC-KC pox vector encoding Env gp140 clade C. Both regimens elicited Env-specific IgG-switched memory hu-B cells at a greater magnitude in hu-mice primed with NYVAC-KC. Single-cell RNA-seq analysis showed gp140-specific hu-B cells to express polyclonal IgG1 and IgG3 isotypes and a broad Ig VH/VL repertoire, with predominant VH3 family gene usage. These cells exhibited a higher rate of somatic hypermutation than the non-specific IgG+ hu-B-cell counterpart. Both vaccine regimens induced splenic GC-like structures containing hu-B and hu-Tfh-like cells expressing PD-1 and BCL-6. We confirmed in this model that circulating ICOS+ memory hu-Tfh cells correlated with the magnitude of gp140-specific B-cell responses. Finally, the NYVAC-KC heterologous prime led to a more diverse clonal expansion of specific hu-B cells. Thus, this study shows that CD40-targeted vaccination induces human IgG production in hu-mice and provides insights for the development of a CD40-targeting vaccine to prevent HIV-1 infection in humans.

DC Subsets Regulate Humoral Immune Responses by Supporting the Differentiation of Distinct Tfh Cells.

To determine the contribution of skin DC subsets in the regulation of humoral immunity, we used a well-characterized antigen targeting system to limit antigen availability and presentation to certain skin-derived DC subsets. Here we show that delivery of foreign antigen to steady state Langerhans cells (LCs) and cDC1s through the same receptor (Langerin) led to, respectively, robust vs. minimal-to-null humoral immune response. LCs, unlike cDC1s, supported the formation of germinal center T follicular helper cells (GC-Tfh) antigen dose-dependently and then, likely licensed by these T cells, some of the LCs migrated to the B cell area to initiate B cell responses. Furthermore, we found that the cDC1s, probably through their superior T cell activation capacity, prevented the LCs from inducing GC-Tfh cells and humoral immune responses. We further show that targeted delivery of cytokines to DCs can be used to modulate DC-induced humoral immune responses, which has important therapeutic potential. Finally, we show that human LCs, unlike monocyte-derived DCs, can support GC Tfh generation in an in vitro autologous system; and in agreement with mouse data, we provide evidence in NHP studies that targeting LCs without adjuvants is an effective way to induce antibody responses, but does not trigger CD8+ T cell responses. Our findings suggest that the major limitations of some relatively ineffective vaccines currently in use or in development might be that (1) they are not formulated to specifically target a certain subset of DCs and/or (2) the antigen dose is not tailored to maximize the intrinsic/pre-programmed capabilities of the specific DC subset. This new and substantial departure from the status quo is expected to overcome problems that have hindered our ability to generate effective vaccines against some key pathogens.

Human innate responses and adjuvant activity of TLR ligands in vivo in mice reconstituted with a human immune system.

TLR ligands (TLR-Ls) represent a class of novel vaccine adjuvants. However, their immunologic effects in humans remain poorly defined in vivo. Using a humanized mouse model with a functional human immune system, we investigated how different TLR-Ls stimulated human innate immune response in vivo and their applications as vaccine adjuvants for enhancing human cellular immune response. We found that splenocytes from humanized mice showed identical responses to various TLR-Ls as human PBMCs in vitro. To our surprise, various TLR-Ls stimulated human cytokines and chemokines differently in vivo compared to that in vitro. For example, CpG-A was most efficient to induce IFN-α production in vitro. In contrast, CpG-B, R848 and Poly I:C stimulated much more IFN-α than CpG-A in vivo. Importantly, the human innate immune response to specific TLR-Ls in humanized mice was different from that reported in C57BL/6 mice, but similar to that reported in nonhuman primates. Furthermore, we found that different TLR-Ls distinctively activated and mobilized human plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs) and monocytes in different organs. Finally, we showed that, as adjuvants, CpG-B, R848 and Poly I:C can all enhance antigen specific CD4+ T cell response, while only R848 and Poly I:C induced CD8+ cytotoxic T cells response to a CD40-targeting HIV vaccine in humanized mice, correlated with their ability to activate human mDCs but not pDCs. We conclude that humanized mice serve as a highly relevant model to evaluate and rank the human immunologic effects of novel adjuvants in vivo prior to testing in humans.

Targeting dendritic cells in humanized mice receiving adoptive T cells via monoclonal antibodies fused to Flu epitopes.

The targeting of vaccine antigens to antigen presenting cells (APC), such as dendritic cells (DCs), is a promising strategy for boosting vaccine immunogenicity and, in turn, protective and/or therapeutic efficacy. However, in vivo systems are needed to evaluate the potential of this approach for testing human vaccines. To this end, we examined human CD8(+) T-cell expansion to novel DC-targeting vaccines in vitro and in vivo in humanized mice. Vaccines incorporating the influenza matrix protein-1 (FluM1) antigen fused to human specific antibodies targeting different DC receptors, including DEC-205, DCIR, Dectin-1, and CD40, elicited human CD8(+) T-cell responses, as defined by the magnitude of specific CD8(+) T-cells to the targeted antigen. In vitro we observed differences in response to the different vaccines, particularly between the weakly immunogenic DEC-205-targeted and more strongly immunogenic CD40-targeted vaccines, consistent with previous studies. However, in humanized mice adoptively transferred (AT) with mature human T cells (HM-T), vaccines that performed weakly in vitro (i.e., DEC-205, DCIR, and Dectin-1) gave stronger responses in vivo, some resembling those of the strongly immunogenic CD40-targeted vaccine. These results demonstrate the utility of the humanized mouse model as a platform for studies of human vaccines.

Skin dendritic cells induce follicular helper T cells and protective humoral immune responses.

BACKGROUND: The contribution of individual subsets of dendritic cells (DCs) to generation of adaptive immunity is central to understanding immune homeostasis and protective immune responses. OBJECTIVE: We sought to define functions for steady-state skin DCs. METHODS: We present an approach in which we restrict antigen presentation to individual DC subsets in the skin and monitor the effects on endogenous antigen-specific CD4(+) T- and B-cell responses. RESULTS: Presentation of foreign antigen by Langerhans cells (LC) in the absence of exogenous adjuvant led to a large expansion of T follicular helper (TFH) cells. This was accompanied by B-cell activation, germinal center formation, and protective antibody responses against influenza. The expansion of TFH cells and antibody responses could be elicited by both systemic and topical skin immunization. TFH cell induction was not restricted to LCs and occurred in response to antigen presentation by CD103(+) dermal DCs. CD103(+) DCs, despite inducing similar TFH responses as LCs, were less efficient in induction of germinal center B cells and humoral immune responses. We also found that skin DCs are sufficient to expand CXCR5(+) TFH cells through an IL-6- and IFN-α/ß receptor-independent mechanism, but B cells were required for sustained Bcl-6(+) expression. CONCLUSIONS: These data demonstrate that a major unappreciated function of skin DCs is their promotion of TFH cells and humoral immune responses that potentially represent an efficient approach for vaccination.

Candida albicans morphology and dendritic cell subsets determine T helper cell differentiation.

Candida albicans is a dimorphic fungus responsible for chronic mucocutaneous and systemic infections. Mucocutaneous immunity to C. albicans requires T helper 17 (Th17) cell differentiation that is thought to depend on recognition of filamentous C. albicans. Systemic immunity is considered T cell independent. Using a murine skin infection model, we compared T helper cell responses to yeast and filamentous C. albicans. We found that only yeast induced Th17 cell responses through a mechanism that required Dectin-1-mediated expression of interleukin-6 (IL-6) by Langerhans cells. Filamentous forms induced Th1 without Th17 cell responses due to the absence of Dectin-1 ligation. Notably, Th17 cell responses provided protection against cutaneous infection while Th1 cell responses provided protection against systemic infection. Thus, C. albicans morphology drives distinct T helper cell responses that provide tissue-specific protection. These findings provide insight into compartmentalization of Th cell responses and C. albicans pathogenesis and have critical implications for vaccine strategies.

Immunologic characterization of a rhesus macaque H1N1 challenge model for candidate influenza virus vaccine assessment.

Despite the availability of annually formulated vaccines, influenza virus infection remains a worldwide public health burden. Therefore, it is important to develop preclinical challenge models that enable the evaluation of vaccine candidates while elucidating mechanisms of protection. Here, we report that naive rhesus macaques challenged with 2009 pandemic H1N1 (pH1N1) influenza virus do not develop observable clinical symptoms of disease but develop a subclinical biphasic fever on days 1 and 5 to 6 postchallenge. Whole blood microarray analysis further revealed that interferon activity was associated with fever. We then tested whether type I interferon activity in the blood is a correlate of vaccine efficacy. The animals immunized with candidate vaccines carrying hemagglutinin (HA) or nucleoprotein (NP) exhibited significantly reduced interferon activity on days 5 to 6 postchallenge. Supported by cellular and serological data, we conclude that blood interferon activity is a prominent marker that provides a convenient metric of influenza virus vaccine efficacy in the subclinical rhesus macaque model.

Systems scale interactive exploration reveals quantitative and qualitative differences in response to influenza and pneumococcal vaccines.

Systems immunology approaches were employed to investigate innate and adaptive immune responses to influenza and pneumococcal vaccines. These two non-live vaccines show different magnitudes of transcriptional responses at different time points after vaccination. Software solutions were developed to explore correlates of vaccine efficacy measured as antibody titers at day 28. These enabled a further dissection of transcriptional responses. Thus, the innate response, measured within hours in the peripheral blood, was dominated by an interferon transcriptional signature after influenza vaccination and by an inflammation signature after pneumococcal vaccination. Day 7 plasmablast responses induced by both vaccines was more pronounced after pneumococcal vaccination. Together, these results suggest that comparing global immune responses elicited by different vaccines will be critical to our understanding of the immune mechanisms underpinning successful vaccination.

Targeting concatenated HIV antigens to human CD40 expands a broad repertoire of multifunctional CD4+ and CD8+ T cells.

OBJECTIVE: Targeting HIV antigens directly to dendritic cells using monoclonal antibodies against cell-surface receptors has been shown to evoke potent cellular immunity in animal models. The objective of this study was to configure an anti-human CD40 antibody fused to a string of five highly conserved CD4 and CD8 T-cell epitope-rich regions of HIV-1 Gag, Nef and Pol (αCD40.HIV5pep), and then to demonstrate the capacity of this candidate therapeutic vaccine to target these HIV peptide antigens to human dendritic cells to expand functional HIV-specific T cells. METHODS: Antigen-specific cytokine production using intracellular flow cytometry and multiplex bead-based assay, and suppression of viral inhibition, were used to characterize the T cells expanded by αCD40.HIV5pep from HIV-infected patient peripheral blood mononuclear cell (PBMC) and dendritic cell/T-cell co-cultures. RESULTS: This candidate vaccine expands memory CD4 and CD8 T cells specific to multiple epitopes within all five peptide regions across a wide range of major histocompatibility complex (MHC) haplotypes from HIV-infected patient PBMC and dendritic cell/T-cell co-cultures. These in vitro expanded HIV antigen-specific CD4 and CD8 T cells produce multiple cytokines and chemokines. αCD40.HIV5pep-expanded CD8 T cells have characteristics of cytotoxic effector cells and are able to kill autologous target cells and suppress HIV-1 replication in vitro. CONCLUSION: Our data demonstrate the therapeutic potential of this CD40-targeting HIV candidate vaccine in inducing a broad repertoire of multifunctional T cells in patients.

Skin-resident murine dendritic cell subsets promote distinct and opposing antigen-specific T helper cell responses.

Skin-resident dendritic cells (DCs) are well positioned to encounter cutaneous pathogens and are required for the initiation of adaptive immune responses. There are at least three subsets of skin DC- Langerhans cells (LC), Langerin(+) dermal DCs (dDCs), and classic dDCs. Whether these subsets have distinct or redundant function in vivo is poorly understood. Using a Candida albicans skin infection model, we have shown that direct presentation of antigen by LC is necessary and sufficient for the generation of antigen-specific T helper-17 (Th17) cells but not for the generation of cytotoxic lymphocytes (CTLs). In contrast, Langerin(+) dDCs are required for the generation of antigen specific CTL and Th1 cells. Langerin(+) dDCs also inhibited the ability of LCs and classic DCs to promote Th17 cell responses. This work demonstrates that skin-resident DC subsets promote distinct and opposing antigen-specific responses.

Human dendritic cells induce the differentiation of interleukin-21-producing T follicular helper-like cells through interleukin-12.

T follicular helper (Tfh) cells help development of antibody responses via interleukin-21 (IL-21). Here we show that activated human dendritic cells (DCs) induced naive CD4(+) T cells to become IL-21-producing Tfh-like cells through IL-12. CD4(+) T cells primed with IL-12 induced B cells to produce immunoglobulins in a fashion dependent on IL-21 and inducible costimulator (ICOS), thus sharing fundamental characteristics with Tfh cells. The induction of Tfh-like cells by activated DCs was inhibited by neutralizing IL-12. IL-12 induced two different IL-21-producers: IL-21(+)IFN-gamma(+)T-bet(+) Th1 cells and IL-21(+)IFN-gamma(-)T-bet(-) non-Th1 cells, in a manner dependent on signal transducer and activator of transcription 4 (STAT4). IL-12 also regulated IL-21 secretion by memory CD4(+) T cells. Thus, IL-12 produced by activated DCs regulates antibody responses via developing IL-21-producing Tfh-like cells and inducing IL-21 secretion from memory CD4(+) T cells. These data suggest that the developmental pathway of Tfh cells differs between mice and humans, which have considerable implications for vaccine development.

The CD200 receptor is a novel and potent regulator of murine and human mast cell function.

CD200R is a member of the Ig supergene family that is primarily expressed on myeloid cells. Recent in vivo studies have suggested that CD200R is an inhibitory receptor capable of regulating the activation threshold of inflammatory immune responses. Here we provide definitive evidence that CD200R is expressed on mouse and human mast cells and that engagement of CD200R by agonist Abs or ligand results in a potent inhibition of mast cell degranulation and cytokine secretion responses. CD200R-mediated inhibition of FcepsilonRI activation was observed both in vitro and in vivo and did not require the coligation of CD200R to FcepsilonRI. Unlike the majority of myeloid inhibitory receptors, CD200R does not contain a phosphatase recruiting inhibitory motif (ITIM); therefore, we conclude that CD200R represents a novel and potent inhibitory receptor that can be targeted in vivo to regulate mast cell-dependent pathologies.

OSCAR is an FcRgamma-associated receptor that is expressed by myeloid cells and is involved in antigen presentation and activation of human dendritic cells.

We have isolated a novel cell surface molecule, the human homolog of osteoclast-associated receptor (OSCAR). Unlike mouse OSCAR, hOSCAR is widely transcribed in cells of the myeloid lineage. Notably, hOSCAR is expressed on circulating blood monocytes and CD11c(+) dendritic cells but not on T and B cells. hOSCAR is continually expressed during differentiation of CD14(+) monocytes into dendritic cells and maintained after maturation. hOSCAR associates with the FcRgamma as shown by translocation of FcRgamma to the cell surface in presence of hOSCAR and coimmunoprecipitation from transfected cell lines and ex vivo cells. Engagement of hOSCAR with specific mAb leads to Ca(2+) mobilization and cytokine release, indicators of cellular activation. Endocytosis of the receptor in dendritic cells was observed, followed by passage of the internalized material into Lamp-1(+) and HLA-DR(+) compartments, suggesting a role in antigen uptake and presentation. Dendritic cells were able to stimulate a T-cell clone specific for an epitope of mouse IgG1 after uptake and processing of the hOSCAR-specific antibody, demonstrating the capacity of this receptor to mediate antigen presentation. hOSCAR thus represents a novel class of molecule expressed by dendritic cells involved in the initiation of the immune response.

Development of a Chinese hamster ovary cell line for recombinant adenovirus-mediated gene expression.

Recombinant human adenovirus (rhAd) has been used extensively for functional protein expression in mammalian cells including those of human and nonhuman origin. High-level protein production by rhAd vectors is expected in their permissive host cells, such as the human embryonic kidney 293 (HEK293) cell line. This is attributed primarily to the permissiveness of HEK293 to rhAd infection and their ability to support viral DNA replication by providing the missing El proteins. However, the HEK293 cells tend to suffer from cytopathic effect (CPE) as a result of virus replication. Under these circumstances, the host cell function is compromised and the culture viability will be reduced. Consequently, newly synthesized polypeptides may not be processed properly at posttranslational levels. Therefore, the usefulness of HEK293 cells for the expression of complex targets such as secreted proteins could be limited. In the search for a more robust cell line as a production host for rhAd expression vectors, a series of screening experiments was performed to isolate clones from Chinese hamster ovary-K1 (CHO-K1) cells. First, multiple rounds of infection of CHO-K1 cells were performed utilizing an rhAd expressing GFP. After each cycle of infection, a small population of CHO cells with high GFP levels was enriched by FACS. Second, individual clones more permissive to human adenovirus infection were isolated from the highly enriched subpopulation by serial dilution. A single clone, designated CHO-K1-C5, was found to be particularly permissive to rhAd infection than the parental pool and has served as a production host in the successful expression of several secreted proteins.

New IL-17 family members promote Th1 or Th2 responses in the lung: in vivo function of the novel cytokine IL-25.

We have biologically characterized two new members of the IL-17 cytokine family: IL-17F and IL-25. In contrast to conventional in vitro screening approaches, we have characterized the activity of these new molecules by direct in vivo analysis and have compared their function to that of other IL-17 family members. Intranasal administration of adenovirus expressing IL-17, IL-17C, or IL-17F resulted in bronchoalveolar lavage neutrophilia and inflammatory gene expression in the lung. In contrast, intranasal administration of IL-25-expressing adenovirus or IL-25 protein resulted in the production of IL-4, IL-5, IL-13, and eotaxin mRNA in the lung and marked eosinophilia in the bronchoalveolar lavage and lung tissue. Mice given intranasal IL-25 also developed epithelial cell hyperplasia, increased mucus secretion, and airway hyperreactivity. IL-25 gene expression was detected following Aspergillus and Nippostrongylus infection in the lung and gut, respectively. IL-25-induced eosinophilia required IL-5 and IL-13, but not IL-4 or T cells. Following IL-25 administration, the IL-5(+) staining cells were CD45R/B220(+), Thy-1(+/-), but were NK1.1-, Ly-6G(GR-1)-, CD4-, CD3-, and c-kit-negative. gamma-common knockout mice did not develop eosinophilia in response to IL-25, nor were IL-5(+) cells detected. These findings suggest the existence of a previously unrecognized cell population that may initiate Th2-like responses by responding to IL-25 in vivo. Further, these data demonstrate the heterogeneity of function within the IL-17 cytokine family and suggest that IL-25 may be an important mediator of allergic disease via production of IL-4, IL-5, IL-13, and eotaxin.