The spike protein (S) of SARS-CoV-2 induces neutralizing antibodies and is the key component of current COVID-19 vaccines. The most efficacious COVID-19 vaccines are genetically-encoded spikes with a ...double proline substitution in the hinge region to stabilize S in the prefusion conformation (S-2P). A subunit vaccine can be a valuable addition to mRNA and viral vector-based vaccines but requires high stability of spike. In addition, further stabilization of the prefusion conformation of spike might improve immunogenicity. To test this, five spike proteins were designed and characterized, ranging from low to high stability. The immunogenicity of these proteins was assessed in mice, demonstrating that a spike (S-closed-2) with a high melting temperature, which still allowed ACE2 binding, induced the highest neutralization titers against homologous and heterologous strains (up to 16-fold higher than the least stabilized spike). In contrast, the most stable spike variant (S-locked), in which the receptor binding domains (RBDs) were locked in a closed conformation and thus not able to breathe, induced relatively low neutralizing antibody titers against heterologous strains. These data demonstrate that S protein stabilization with RBDs exposing highly conserved epitopes may be needed to increase the immunogenicity of spike proteins for future COVID-19 vaccines.
Dendritic cells (DCs) are central for the induction of T cell immunity and tolerance. Fundamental for DCs to control the immune system is their differentiation from precursors into various DC subsets ...with distinct functions and locations in lymphoid organs and tissues. In contrast to the differentiation of epidermal Langerhans cells (LCs) and their seeding into the epidermis, LC maturation, turnover, and MHC class II Ag presentation capacities are strictly dependent on the presence of Dicer, which generates mature microRNAs (miRNAs). Absence of miRNAs caused a strongly disturbed steady-state homeostasis of LCs by increasing their turnover and apoptosis rate, leading to progressive ablation of LCs with age. The failure to maintain LCs populating the epidermis was accompanied by a proapoptotic gene expression signature. Dicer-deficient LCs showed largely increased cell sizes and reduced expression levels of the C-type lectin receptor Langerin, resulting in the lack of Birbeck granules. In addition, LCs failed to properly upregulate MHC class II, CD40, and CD86 surface molecules upon stimulation, which are critical hallmarks of functional DC maturation. This resulted in inefficient induction of CD4 T cell proliferation, whereas Dicer-deficient LCs could properly stimulate CD8 T cells. Taken together, Dicer-dependent generation of miRNAs affects homeostasis and function of epidermal LCs.
Durable protection against complex pathogens is likely to require immunity that comprises both humoral and cellular responses. While heterologous prime-boost regimens based on recombinant, ...replication-incompetent Adenoviral vectors (AdV) and adjuvanted protein have been able to induce high levels of concomitant humoral and cellular responses, complex manufacturing and handling in the field may limit their success. To combine the benefits of genetic and protein-based vaccination within one vaccine construct and to facilitate their use, we generated Human Adenovirus 35 (HAdV35) vectors genetically encoding a model antigen based on the Plasmodium falciparum (P. falciparum) circumsporozoite (CS) protein and displaying a truncated version of the same antigen (CSshort) via protein IX on the capsid, with or without a flexible glycine-linker and/or a 45Å-spacer. The four tested pIX-antigen display variants were efficiently incorporated and presented on the HAdV35 capsid irrespective of whether a transgene was encoded or not. Transgene-expression and producibility of the display-/expression vectors were not impeded by the pIX-display. In mice, the pIX-modified vectors induced strong humoral antigen-specific immunity that increased with the inclusion of the linker-/spacer molecules, exceeded the responses induced by the genetic, transgene-expressing HAdV35 vector, and surpassed recombinant protein in potency. In addition, the pIX- display/expression vectors elicited high antigen-specific cellular immune responses that matched those of the genetic HAdV35 vector expressing CS. pIX-modified display-/expression HAdV vectors may therefore be a valuable technology for the development of vaccines against complex pathogens, especially in resource-limited settings.
microRNAs have emerged as a novel layer of regulation of cellular development and function, including cells of the immune system. microRNA expression profiles and function of several microRNAs have ...been elucidated in granulocyte macrophage colony-stimulating factor derived dendritic cells (GM-CSF DC). In this study we determined the microRNA expression profile from plasmacytoid DC (pDC) and conventional DC (cDC) generated in murine FMS-related tyrosine kinase 3 ligand (Flt3L) bone marrow culture. We observed distinct miRNA expression signatures in these two different DC subsets and found that pDC were closer related to CD4
+ T cells than to cDC. Expression of a selected subset of microRNAs was also compared between cDC and GM-CSF DC. Furthermore, we show that inhibition of two differentially expressed microRNAs, miR-221 and miR-222, during differentiation resulted in skewed pDC/cDC ratios. Among the confirmed or potential targets for miR-221 and miR-222 are c-Kit, p27
kip1 and E2-2. While c-Kit is expressed by DC progenitors and p27
kip1 is a cell cycle regulator, E2-2 does transcriptionally regulate pDC development. Our data demonstrate that microRNAs can influence Flt3-driven DC differentiation.
Correlates of protection (CoP) are invaluable for iterative vaccine design studies, especially in pursuit of complex vaccines such as a universal influenza vaccine (UFV) where a single antigen is ...optimized to elicit broad protection against many viral antigenic variants. Since broadly protective antibodies against influenza virus often exhibit mutational evidence of prolonged diversification, we studied germinal center (GC) kinetics in hemagglutinin (HA) immunized mice. Here we report that as early as 4 days after secondary immunization, the expansion of HA-specific GC B cells inversely correlated to protection against influenza virus challenge, induced by the antigen. In contrast, follicular T helper (TFH) cells did not expand differently after boost vaccination, suggestive of a B-cell intrinsic difference in activation and differentiation inferred by protective antigen properties. Importantly, differences in antigen dose only affected GC B-cell frequencies after primary immunization. The absence of accompanying differences in total anti-HA or epitope-specific antibody levels induced by vaccines of different efficacy suggests that the GC B-cell response upon revaccination represents an early and unique marker of protection that may significantly accelerate the pre-clinical phase of vaccine development.
Seasonal influenza vaccines are updated almost annually to match the antigenic drift in influenza hemagglutinin (HA) surface glycoprotein. A new HA stem-based antigen, the so-called "mini-HA," was ...recently shown to induce cross-protective antibodies. However, cross-reactive antibodies targeting the HA stem can also be found in mice and humans after administration of seasonal vaccine. This has raised the question whether in similar conditions such a mini-HA would be able to show an increased breadth of protection over immunization with full length (FL) HA. We show in mice that in a direct comparison to H1 FL HA, using the same immunization regimen, dosing and adjuvant, a group 1 mini-HA has a higher protective efficacy against group 1 influenza virus challenges not homologous to the H1 FL HA. Although both antigens induce a similar breadth of HA subtype binding, mini-HA immunization induces significantly more HA stem-specific antibodies correlating with survival. In addition, both mini-HA and H1 FL HA immunization induce influenza neutralizing antibodies while mini-HA induces significantly higher levels of mFcγRIII activation, involved in Fc-mediated antibody effector functions. In agreement with previous findings, this confirms that more than one mechanism contributes to protection against influenza. Together our results further warrant the development of a universal influenza vaccine based on the HA stem region.
Timing of TLR ligation and facilitated antigen uptake dictate the potential of pDCs to present endogenous or exogenous Ag by influencing endosomal traffic and antigen processing machinery.
Subsets of ...antigen‐presenting cDCs have a differential capacity to present exogenous and endogenous protein antigens to CD4+ and/or CD8+ T lymphocytes, depending on expression of antigen‐uptake receptors, processing machinery, and microbial instruction. pDCs are also capable of antigen presentation, but the conditions under which they do this have not been systematically addressed. Highly purified cDCs and pDCs were exposed to exogenous, soluble OVA peptide or whole protein. Alternatively, they were made to express cytoplasmic or endosomal OVA by retroviral transduction or by infection with influenza virus containing OVA epitopes. Like cDCs, pDCs expressed the MHC I processing machinery and could present endogenous or cross‐present exogenous OVA to CD8+ T cells, provided they had been stimulated by CpG motif TLR9 ligands or by influenza. Unlike cDCs, the cross‐priming activity of pDCs was enhanced, not decreased, by simultaneous TLR stimulation. Processing and presentation of exogenous OVA to CD4+ T cells required TLR9 ligation prior to antigen encounter and addition of OVA‐specific Igs. These stimuli up‐regulated critical MHC II processing machinery and enhanced routing to acidic endosomal organelles in a FcγRII‐dependent manner. Endogenous antigen was not presented to CD4+ T cells when expressed in the cytoplasm of pDCs by retrovirus or contained in influenza, unless an Ii‐chain‐derived endosomal routing signal was present. Thus, timing of TLR ligation and facilitated antigen uptake dictate the potential of pDCs to present endogenous or exogenous antigen by influencing endosomal traffic and antigen‐processing machinery.
Seasonal influenza vaccines must be updated annually and suboptimally protect against strains mismatched to the selected vaccine strains. We previously developed a subunit vaccine antigen consisting ...of a stabilized trimeric influenza A group 1 hemagglutinin (H1) stem protein that elicits broadly neutralizing antibodies. Here, we further optimized the stability and manufacturability of the H1 stem antigen (H1 stem v2, also known as INFLUENZA G1 mHA) and characterized its formulation and potency with different adjuvants in vitro and in animal models. The recombinant H1 stem antigen (50 µg) was administered to influenza-naïve non-human primates either with aluminum hydroxide Al(OH)
+ NaCl, AS01
, or SLA-LSQ formulations at week 0, 8 and 34. These SLA-LSQ formulations comprised of varying ratios of the synthetic TLR4 agonist 'second generation synthetic lipid adjuvant' (SLA) with liposomal QS-21 (LSQ). A vaccine formulation with aluminum hydroxide or SLA-LSQ (starting at a 10:25 µg ratio) induced HA-specific antibodies and breadth of neutralization against a panel of influenza A group 1 pseudoviruses, comparable with vaccine formulated with AS01
, four weeks after the second immunization. A formulation with SLA-LSQ in a 5:2 μg ratio contained larger fused or aggregated liposomes and induced significantly lower humoral responses. Broadly HA stem-binding antibodies were detectable for the entire period after the second vaccine dose up to week 34, after which they were boosted by a third vaccine dose. These findings inform about potential adjuvant formulations in clinical trials with an H1 stem-based vaccine candidate.
Mouse models of allergic asthma are increasingly used to study the immunopathology of this complex disorder. The degree and type of airway inflammation is often studied by determination of ...differential cell counts on cytospins of bronchoalveolar lavage fluid (BALF) cells stained with May–Grünwald Giemsa, in which the separation of eosinophils (eos) from neutrophils (neutro) and of monocytes (mono) from activated T cells can be quite problematic. In this study, we compared differential cell counts based on morphological criteria on May–Grünwald Giemsa stained cytospins with a newly developed flow cytometric method. BAL fluid cells were identified based on forward and side scatter characteristics (FSC and SSC), autofluorescence of macrophages, and simultaneous one-step staining with antibodies for T cells (CD3-Cy-Chrome), B cells (B220-Cy-Chrome), eosinophils (CCR3-PE), and dendritic cells (DCs) (MHCII-FITC, CD11c-APC). The validity of this flow cytometric determination was tested by morphological analysis of flow-sorted cellular subsets. In an animal model of ovalbumin-induced asthma, this new method correlated very well with the differential counts based on cytospins. Flow cytometric determination of the cellular composition of BAL fluid in mouse models of asthma is a rapid and easy method that can replace differential cell counts based on morphology.
Seasonal vaccines are currently the most effective countermeasure against influenza. However, seasonal vaccines are only effective against strains closely related to the influenza strains contained ...in the vaccine. Recently a new hemagglutinin (HA) stem-based antigen, the so-called "mini-HA", has been shown to induce a cross-protective immune response in influenza-naive mice and non-human primates (NHP). However, prior exposure to influenza can have a profound effect on the immune response to subsequent influenza infection and the protective efficacy of vaccination. Here we show that mini-HA, compared to a trivalent influenza vaccine (TIV), elicits a broadened influenza-specific humoral immune response in NHP previously exposed to influenza. Serum transfer experiments showed that antibodies induced by both mini-HA and seasonal vaccine protected mice against lethal challenge with a H1N1 influenza strain heterologous to the H1 HA included in the TIV. However, antibodies elicited by mini-HA showed an additional benefit of protecting mice against lethal heterosubtypic H5N1 influenza challenge, associated with H5 HA-specific functional antibodies.
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