Adjuvanting a subunit SARS-CoV-2 vaccine with clinically relevant adjuvants induces durable protection in mice.

Adjuvants enhance the magnitude and the durability of the immune response to vaccines. However, there is a paucity of comparative studies on the nature of the immune responses stimulated by leading adjuvant candidates. In this study, we compared five clinically relevant adjuvants in mice-alum, AS03 (a squalene-based adjuvant supplemented with α-tocopherol), AS37 (a TLR7 ligand emulsified in alum), CpG1018 (a TLR9 ligand emulsified in alum), O/W 1849101 (a squalene-based adjuvant)-for their capacity to stimulate immune responses when combined with a subunit vaccine under clinical development. We found that all four of the adjuvant candidates surpassed alum with respect to their capacity to induce enhanced and durable antigen-specific antibody responses. The TLR-agonist-based adjuvants CpG1018 (TLR9) and AS37 (TLR7) induced Th1-skewed CD4+ T cell responses, while alum, O/W, and AS03 induced a balanced Th1/Th2 response. Consistent with this, adjuvants induced distinct patterns of early innate responses. Finally, vaccines adjuvanted with AS03, AS37, and CpG1018/alum-induced durable neutralizing-antibody responses and significant protection against the B.1.351 variant 7 months following immunization. These results, together with our recent results from an identical study in non-human primates (NHPs), provide a comparative benchmarking of five clinically relevant vaccine adjuvants for their capacity to stimulate immunity to a subunit vaccine, demonstrating the capacity of adjuvanted SARS-CoV-2 subunit vaccines to provide durable protection against the B.1.351 variant. Furthermore, these results reveal differences between the widely-used C57BL/6 mouse strain and NHP animal models, highlighting the importance of species selection for future vaccine and adjuvant studies.

The single-cell epigenomic and transcriptional landscape of immunity to influenza vaccination.

Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant.

Adjuvanting a subunit COVID-19 vaccine to induce protective immunity.

The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).

Safety of AS03-adjuvanted influenza vaccines: A review of the evidence.

Clinical and post-licensure data have demonstrated that AS03-adjuvanted inactivated split virion vaccines, many with reduced antigen content, are effective against influenza infection. The objective of this review is to provide a comprehensive assessment of the safety of trivalent seasonal, monovalent pre-pandemic and pandemic AS03-adjuvanted influenza vaccines, based on non-clinical, clinical and post-licensure data in various populations. Non-clinical studies on local tolerance, toxicology and safety pharmacology did not raise any safety concerns with AS03 administered alone or combined with various influenza antigens. Data from clinical trials with over 55,000 vaccinated subjects showed that AS03-adjuvanted influenza vaccines were generally well tolerated and displayed an acceptable safety profile, although the power to detect rare events was limited. Approximately 90 million doses of A/H1N1pdm09 pandemic influenza vaccines (Pandemrix and Arepanrix H1N1) were administered worldwide, which contributed post-licensure data to the collective safety data for AS03-adjuvanted influenza vaccines. An association between Pandemrix and narcolepsy was observed during the A/H1N1pdm09 pandemic, for which a role of a CD4 T cell mimicry sequence in the haemagglutinin protein of A/H1N1pdm09 cannot be excluded. Provided that future AS03-adjuvanted influenza vaccines do not contain this putative mimicry sequence, this extensive safety experience supports the further development and use of AS03-adjuvanted inactivated split virion candidate vaccines against seasonal and pandemic influenza infections.

Long-Term Persistence of Cell-Mediated and Humoral Responses to A(H1N1)pdm09 Influenza Virus Vaccines and the Role of the AS03 Adjuvant System in Adults during Two Randomized Controlled Trials.

We investigated the role of AS03A (here AS03), an α-tocopherol oil-in-water emulsion-based adjuvant system, on the long-term persistence of humoral and cell-mediated immune responses to A(H1N1)pdm09 influenza vaccines. In two studies, a total of 261 healthy adults (≤60 years old) were randomized to receive two doses of AS03-adjuvanted vaccine containing 3.75 µg of hemagglutinin (HA) or nonadjuvanted vaccine containing 15 µg of hemagglutinin (in study A) or 3.75 µg of hemagglutinin (in study B) 21 days apart. Hemagglutination inhibition (HI) antibody, memory B-cell, and CD4+/CD8+ T-cell responses were characterized up to 1 year following dose 1. We also assessed the effects of age and seasonal influenza vaccination history. AS03-adjuvanted (3.75 µg HA) vaccine and nonadjuvanted vaccine at 15 µg but not at 3.75 µg HA elicited HI antibody responses persisting at levels that continued to meet European licensure criteria through month 12. At month 12, the geometric mean titer for AS03-adjuvanted vaccine was similar to that for nonadjuvanted (15-µg) vaccine in study A (1:86 and 1:88, respectively) and higher than that for nonadjuvanted (3.75-µg) vaccine in study B (1:77 and 1:35, respectively). A(H1N1)pdm09-specific CD4+ T-cell and B-cell responses were stronger in AS03-adjuvanted groups and persisted only in these groups for 12 months at levels exceeding prevaccination frequencies. Advancing age and a seasonal vaccination history tended to reduce HI antibody and memory B-cell responses and, albeit less consistently, CD4+ T-cell responses. Thus, AS03 seemed to enhance the persistence of humoral and cell-mediated responses to A(H1N1)pdm09 vaccine, allowing for antigen sparing and mitigating potential negative effects of age and previous seasonal vaccination. (These studies have been registered at ClinicalTrials.gov under registration no. NCT00968539 and NCT00989287.).

Priming with AS03 A-adjuvanted H5N1 influenza vaccine improves the kinetics, magnitude and durability of the immune response after a heterologous booster vaccination: an open non-randomised extension of a double-blind randomised primary study.

An influenza vaccine with cross-immunogenic potential could play a key role in pandemic mitigation by promoting a rapid immune response to infection and/or subsequent vaccination with strains drifted from the primary vaccine strain. Here we assess the role of AS03(A) (an oil-in-water emulsion based Adjuvant System containing tocopherol) in this prime-boost concept using H5N1 as a model shift influenza antigen. In this open, non-randomised study (NCT00506350; an extension of an earlier randomised study) we assessed immunogenicity in nine groups of 35-50 volunteers aged 19-61 years following administration of AS03(A)-adjuvanted split-virion H5N1 vaccine containing 3.75mug of haemagglutinin (HA) from the A/Indonesia/5/2005(IBCDC-RG2) clade 2.1 strain. A single booster dose of vaccine was administered to four groups primed 14 months previously with different HA levels of AS03(A)-adjuvanted clade 1 A/Vietnam/1194/2004 H5N1 vaccine. Two booster doses (given 21 days apart) were administered to four groups primed 14 months previously with different HA levels of non-adjuvanted A/Vietnam/1194/2004 H5N1 vaccine and also to a control group of un-primed subjects. In individuals primed 14 months earlier with AS03(A)-adjuvanted A/Vietnam/1194/2004 vaccines, a single booster dose of AS03(A)-adjuvanted A/Indonesia/5/2005 induced rapid immune responses (licensure criteria met in 7-14 days) comparable to that observed in the un-primed control group following two doses of adjuvanted vaccine. In contrast, individuals primed with non-adjuvanted formulations exhibited minimal immune responses which, even after two doses, were unexpectedly much lower than that observed in un-primed subjects. AS03(A) enhances the initial priming effect of pandemic influenza vaccination enabling a rapid humoral response to single dose boosting with a heterologous strain at 14 months. In contrast, priming without adjuvant appears to inhibit the response to subsequent vaccination with a heterologous strain. These findings should guide the development of vaccines to combat the present influenza A/H1N1 pandemic.

Antitumor efficacy of the novel chemotherapeutic agent coramsine is potentiated by cotreatment with CpG-containing oligodeoxynucleotides.

Coramsine is a novel chemotherapeutic agent isolated from Solanum linnaeanum (devil's apple). Topical treatment provides clinical benefit for skin tumors. To evaluate the potential broader applicability of the drug, its in vivo anticancer efficacy in a murine model of malignant mesothelioma and its mode of action were investigated. Systemic administration of coramsine slowed tumor growth and prolonged survival time. Importantly, the antitumor efficacy of coramsine was enhanced when treatment was combined with stimulation of innate immunity using unmethylated CpG-containing oligodeoxynucleotides (ODNs). Combination treatment further slowed tumor growth and provided a survival benefit. Coramsine seems to kill tumor cells by direct cell lysis. Using 2 different assays to detect apoptosis (caspase activation and DNA fragmentation), we found no evidence that coramsine induces any form of programmed cell death. The fact that the efficacy of coramsine is potentiated by CpG ODNs suggests that coramsine-induced cell death is an immunologic null event.

Fast track selection of immunogens for novel vaccines through visualisation of the early onset of the B-cell response.

A most essential step in vaccine research and development, ie vaccine studies in animals, seriously suffer from long timespans needed to arrive at effective immunogens. In this report we show how almost immediately after vaccination the antibody inducing potential of low immunogenic 'self' antigens can be accurately assessed. (We expect that this timespan can be reduced even more when 'non self' antigens are used, since such responses should be stronger.) The method takes advantage of the immediate onset after vaccination of the immune response in the spleen. This novel method allows detection of antigen-specific B cells of the spleen as early as 7 days after immunization and at frequencies as low as 10 in 1,000,000 cells. The method depends on sequential staining with PE- and APC-conjugated tetramers, made with the same biotinylated peptide. The antigenic peptides are biotinylated and tetramerized with either PE neutravidin or APC streptavidin. We expect that this method can be generally applied to visualize B cell responses, irrespective of the way they are induced. In addition to the fast selection and development of novel immunogens, this procedure can be used to delineate the kinetics of the B cell response, to phenotypically characterize and to isolate antigen-specific B cells, and, perhaps most importantly, to count them at the clonal level before any circulating antibodies can be detected.