Vaccines targeting the conserved stem region of influenza hemagglutinin (HA) may induce broad immunity and reduce reliance on strain-matched seasonal formulations. This Phase 1/2a study evaluates safety and immunogenicity of a universal influenza vaccine component comprising a soluble trimeric stabilized group 1 HA-stem protein (INFLUENZA G1 mHA). Healthy adults aged 18-to-45 received one or two doses at 45 µg or 135 µg, with or without Al(OH)₃. Primary endpoints were safety and reactogenicity. All regimens were well-tolerated with no vaccine-related serious adverse events. Secondary and exploratory endpoints included binding, neutralizing, and ADCC responses compared to a matched quadrivalent seasonal influenza vaccine cohort. A single dose induced robust binding antibody responses to H5 HA and homologous stem HA, with 6.5-16.4-fold increases at Day 29 versus baseline, exceeding responses elicited by the seasonal influenza vaccine. Additionally, we demonstrate broader binding to a panel of group 1 HAs, with responses maintained for at least one year. Neutralizing antibody and ADCC responses at Day 29 showed 7.9-15-fold and 4.9-8.6-fold increases, respectively. Importantly, serum from vaccinated participants protect mice against lethal heterologous H5N1 challenge. This study provides proof of concept that the HA stem vaccine elicits broad and durable influenza A group 1-directed immunity. This trial is registered with ClinicalTrials.gov (NCT05901636).

Human metapneumovirus (HMPV) is a significant respiratory pathogen, particularly in vulnerable populations. Background: No vaccine for the prevention of HMPV is currently licensed, although several subunit vaccines are in development. Saponin-based adjuvant systems (AS), including QS-21, have transformed the field of subunit vaccines by dramatically increasing their potency and efficacy, leading to the development of several licensed vaccines. However, naturally sourced tree bark-extracted QS-21 faces supply and manufacturing challenges, hindering vaccine development. Objective: This study reports on an alternative plant cell culture system for the consistent production of highly pure QS-21. Method: We evaluated the efficacy of cultured plant cell (cpc)-produced QS-21 in a novel HMPV vaccine, formulating a recombinant pre-fusion stabilized HMPV F protein (preF) with cpcQS-21 and a synthetic toll-like receptor 4 (TLR4) agonist adjuvant formulation. Results: In mice, TLR4 agonist containing adjuvant formulations with plant cell-produced QS-21 performed equally to licensed adjuvant AS01 containing tree-bark-extracted QS-21 and demonstrated a significant increase in immunogenicity against HMPV preF compared to the unadjuvanted control. Conclusion: Our findings pave the way for a reliable, scalable, and sustainable source of pure QS-21, enabling the development of highly effective HMPV and other vaccines with significant public health impact.

AbstractNewly approved subunit and mRNA vaccines for respiratory syncytial virus (RSV) demonstrate effectiveness in preventing severe disease, with protection exceeding 80% primarily through the generation of antibodies. An alternative vaccine platform called self-amplifying RNA (saRNA) holds promise in eliciting humoral and cellular immune responses. We evaluate the immunogenicity of a lipid nanoparticle (LNP)-formulated saRNA vaccine called SMARRT.RSV.preF, encoding a stabilized form of the RSV fusion protein, in female mice and in non-human primates (NHPs) that are either RSV-naïve or previously infected. Intramuscular vaccination with SMARRT.RSV.preF vaccine induces RSV neutralizing antibodies and cellular responses in naïve mice and NHPs. Importantly, a single dose of the vaccine in RSV pre-exposed NHPs elicits a dose-dependent anamnestic humoral immune response comparable to a subunit RSV preF vaccine. Notably, SMARRT.RSV.preF immunization significantly increases polyfunctional RSV.F specific memory CD4+ and CD8+ T-cells compared to RSV.preF protein vaccine. Twenty-four hours post immunization with SMARRT.RSV.preF, there is a dose-dependent increase in the systemic levels of inflammatory and chemotactic cytokines associated with the type I interferon response in NHPs, which is not observed with the protein vaccine. We identify a cluster of analytes including IL-15, TNFα, CCL4, and CXCL10, whose levels are significantly correlated with each other after SMARRT.RSV.preF immunization. These findings suggest saRNA vaccines have the potential to be developed as a prophylactic RSV vaccine based on innate, cellular, and humoral immune profiles they elicit.

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.

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)3] + NaCl, AS01B, 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 AS01B, 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.

Since the original outbreak of the SARS-CoV-2 virus, several rapidly spreading SARS-CoV-2 variants of concern (VOC) have emerged. Here, we show that a single dose of Ad26.COV2.S (based on the Wuhan-Hu-1 spike variant) protects against the Gamma and Delta variants in naive hamsters, supporting the observed maintained vaccine efficacy in humans against these VOC. Adapted spike-based booster vaccines targeting Omicron variants have now been authorized in the absence of human efficacy data. We evaluated the immunogenicity and efficacy of Ad26.COV2.S.529 (encoding a stabilized Omicron BA.1 spike) in naive mice and in hamsters with pre-existing immunity to the Wuhan-Hu-1 spike. In naive mice, Ad26.COV2.S.529 elicited higher neutralizing antibody titers against SARS-CoV-2 Omicron BA.1 and BA.2, compared with Ad26.COV2.S. However, neutralizing titers against the SARS-CoV-2 B.1 (D614G) and Delta variants were lower after primary vaccination with Ad26.COV2.S.529 compared with Ad26.COV2.S. In contrast, we found comparable Omicron BA.1 and BA.2 neutralizing titers in hamsters with pre-existing Wuhan-Hu-1 spike immunity after vaccination with Ad26.COV2.S, Ad26.COV2.S.529 or a combination of the two vaccines. Moreover, all three vaccine modalities induced equivalent protection against Omicron BA.2 challenge in these animals. Overall, our data suggest that an Omicron BA.1-based booster in rodents does not improve immunogenicity and efficacy against Omicron BA.2 over an Ad26.COV2.S booster in a setting of pre-existing immunity to SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant sparked concern due to its fast spread and the unprecedented number of mutations in the spike protein that enables it to partially evade spike-based COVID-19 vaccine-induced humoral immunity. In anticipation of a potential need for an Omicron spike-based vaccine, we generated an Ad26 vector encoding an Omicron (BA.1) spike protein (Ad26.COV2.S.529). Ad26.COV2.S.529 encodes for a prefusion stabilized spike protein, similar to the current COVID-19 vaccine Ad26.COV2.S encoding the Wuhan-Hu-1 spike protein. We verified that spike expression by Ad26.COV2.S.529 was comparable to Ad26.COV2.S. Immunogenicity of Ad26.COV2.S.529 was then evaluated in naïve mice and SARS-CoV-2 Wuhan-Hu-1 spike pre-immunized hamsters. In naïve mice, Ad26.COV2.S.529 elicited robust neutralizing antibodies against SARS-CoV-2 Omicron (BA.1) but not to SARS-CoV-2 Delta (B.1.617.2), while the opposite was observed for Ad26.COV2.S. In pre-immune hamsters, Ad26.COV2.S.529 vaccination resulted in robust increases in neutralizing antibody titers against both SARS-CoV-2 Omicron (BA.1) and Delta (B.1.617.2), while Ad26.COV2.S vaccination only increased neutralizing antibody titers against the Delta variant. Our data imply that Ad26.COV2.S.529 can both expand and boost a Wuhan-Hu-1 spike-primed humoral immune response to protect against distant SARS-CoV-2 variants.

Safe and effective coronavirus disease-19 (COVID-19) vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses may improve the magnitude and durability of immunity and protective efficacy. We assessed one- and two-dose regimens of the Ad26.COV2.S vaccine candidate in adult and aged nonhuman primates (NHPs). A two-dose Ad26.COV2.S regimen induced higher peak binding and neutralizing antibody responses compared with a single dose. In one-dose regimens, neutralizing antibody responses were stable for at least 14 wk, providing an early indication of durability. Ad26.COV2.S induced humoral immunity and T helper cell (Th cell) 1-skewed cellular responses in aged NHPs that were comparable to those in adult animals. Aged Ad26.COV2.S-vaccinated animals challenged 3 mo after dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. Neutralization of variants of concern by NHP sera was reduced for B.1.351 lineages while maintained for the B.1.1.7 lineage independent of Ad26.COV2.S vaccine regimen.

The development of preventive corona virus disease (COVID)-19 vaccines is an urgent need, especially for the aging population that is most affected by the ongoing pandemic. The Janssen Ad26.COV2.S vaccine candidate is currently the only one evaluated as a single dose vaccination regimen in Phase 3 clinical studies. While the advantages of single dose vaccines, especially for use during a pandemic, are obvious, multiple doses may potentially improve magnitude and durability of immune responses. Here we assessed the immunogenicity of oneand two-dose Ad26.COV2.S vaccine regimens in adult and aged non-human primates (NHP). A second vaccine dose, administered 8 weeks post the first immunization, induced a significant increase in antigenspecific binding and neutralizing antibody responses in both adult and aged animals as compared to a single dose. In addition, in one-dose regimens neutralizing antibody responses were maintained for at least 14 weeks, providing an early indication of durable immune responses elicited by Ad26.COV2.S. Similar to what we showed previously in adult animals, Ad26.COV2.S vaccination of aged NHP induced a CD8+ T cell response and a Th1 skewed CD4+ T cell response. These data support the initiation of a two-dose Ad26.COV2.S regimen in a Phase 3 clinical trial in adults and elderly.

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 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.

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.

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.

The identification of human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem revitalized hopes of developing a universal influenza vaccine. Using a rational design and library approach, we engineered stable HA stem antigens ("mini-HAs") based on an H1 subtype sequence. Our most advanced candidate exhibits structural and bnAb binding properties comparable to those of full-length HA, completely protects mice in lethal heterologous and heterosubtypic challenge models, and reduces fever after sublethal challenge in cynomolgus monkeys. Antibodies elicited by this mini-HA in mice and nonhuman primates bound a wide range of HAs, competed with human bnAbs for HA stem binding, neutralized H5N1 viruses, and mediated antibody-dependent effector activity. These results represent a proof of concept for the design of HA stem mimics that elicit bnAbs against influenza A group 1 viruses.

ABSTRACT Influenza virus infection of nonhuman primates is a well-established animal model for studying pathogenesis and for evaluating prophylactic and therapeutic intervention strategies. However, usually a standard dose is used for the infection, and there is no information on the relation between challenge dose and virus replication or the induction of immune responses. Such information is also very scarce for humans and largely confined to evaluation of attenuated virus strains. Here, we have compared the effect of a commonly used dose (4 × 10 6 50% tissue culture infective doses) versus a 100-fold-higher dose, administered by intrabronchial installation, to two groups of 6 cynomolgus macaques. Animals infected with the high virus dose showed more fever and had higher peak levels of gamma interferon in the blood. However, virus replication in the trachea was not significantly different between the groups, although in 2 out of 6 animals from the high-dose group it was present at higher levels and for a longer duration. The virus-specific antibody response was not significantly different between the groups. However, antibody enzyme-linked immunosorbent assay, virus neutralization, and hemagglutination inhibition antibody titers correlated with cumulative virus production in the trachea. In conclusion, using influenza virus infection in cynomolgus macaques as a model, we demonstrated a relationship between the level of virus production upon infection and induction of functional antibody responses against the virus. IMPORTANCE There is only very limited information on the effect of virus inoculation dose on the level of virus production and the induction of adaptive immune responses in humans or nonhuman primates. We found only a marginal and variable effect of virus dose on virus production in the trachea but a significant effect on body temperature. The induction of functional antibody responses, including virus neutralization titer, hemagglutination inhibition titer, and antibody-dependent cell-mediated cytotoxicity, correlated with the level of virus replication measured in the trachea. The study reveals a relationship between virus production and functional antibody formation, which could be relevant in defining appropriate criteria for new influenza virus vaccine candidates.

ABSTRACTSubsets 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.

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.

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.

Background. In human lung transplantation, chronic rejection is accompanied by obliterative bronchiolitis (OB), a fibrosing inflammatory condition that leads to occlusion of the bronchial lumen and graft failure. The pathogenesis of this disorder is poorly understood, but likely involves antigen presentation by dendritic cells (DC). We studied the presence and activation status of DCs in transplanted tracheas in a mouse model of OB and studied the effect on graft luminal patency of blocking the costimulatory B7RP-1/inducible costimulator (ICOS) pathway. Methods. Tracheas from Balb/C or from C57Bl/6 mice were transplanted heterotopically under the dorsal skin of C57Bl/6 mice. Histologic, fluorescence-activated cell sorter, and quantitative-polymerase chain reaction analyses were performed after 1, 2, or 4 weeks. In some groups, treatment with blocking rat anti-mICOS antibodies or irrelevant rat immunoglobulin G was administered during the entire observation period. Results. After heterotopic transplantation, both CD103+CD11b− and CD103−CD11b+MHC II+DCs accumulated in the airway epithelium as early as 1 week after allogeneic (mismatched) but not syngeneic (matched) transplantation. Four weeks after Tx, infiltration with CD11c+ MHCII+DCs and CD8+ lymphocytes, luminal fibrosis and epithelial damage were more pronounced in the allogeneic than in the syngeneic setting. There was a 10-fold up-regulation of ICOS mRNA and of chemokines involved in T-cell influx in the mismatched setting compared with the matched setting. Strikingly, anti-ICOS treatment without other immunosuppression prevented luminal fibrosis in mismatched transplants. Conclusions. Our results suggest that early infiltration by DC occurs in posttransplant OB. Blocking critical costimulatory molecules expressed on DCs, as in the B7RP1-ICOS pathway, prevents epithelial damage and luminal fibrosis.