Abstract
Current influenza vaccines face challenges due to antigenic evolution of the circulating virus and waning immunity in humans. Here we investigated the durability of humoral immunity induced by an influenza vaccine based on AS03-adjuvanted chimeric hemagglutinin (cHA) in nonhuman primates (NHPs). Two groups of NHPs received two doses of a seasonal quadrivalent influenza vaccine, followed by sequential immunization with split virus cHA vaccines cH8/1N1, and cH5/1N1. One group received cHA immunizations with AS03 adjuvant. We monitored serum antibodies and long-lived plasma cells in bone marrow for nearly 2 years after the final vaccination. cHA vaccines induced stalk-specific antibody responses. The addition of AS03 enhanced both the magnitude and durability of humoral immunity by establishing long-lived plasma cells in the bone marrow and lymph nodes for nearly 2 years. Passive transfer of NHP serum provided protection against challenge with heterologous influenza A virus strains in mice. This study highlights the potential of the AS03-adjuvanted chimeric HA vaccine strategy to provide durable and broadly protective humoral immunity.
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Influenza is one of the major causes of global morbidity and mortality. Despite the availability of vaccines, an alarming 3–5 million individuals suffer severe illness annually, with 290,000 to 650,000 succumbing to influenza-related respiratory illnesses1. The seasonal influenza vaccine effectiveness in the United States has been reported to vary from 20% to 60% over the last 14 years2. Influenza viruses evolve over time due to antigenic drift3,4. Therefore, the influenza vaccine needs to be matched every season to the circulating strains. Strain selection is based on surveillance and predictions, and mismatches occur that can lead to low vaccine effectiveness. In addition, current seasonal influenza virus vaccines are unlikely to protect from future pandemic influenza virus strains. Thus, there is a great need for the development of a universal influenza vaccine that induces durable and broadly cross-reactive immunity for many years.
Hemagglutinin (HA) is the most abundant glycoprotein present on the surface of influenza virus5. It is the key antigen involved in the induction of immune responses and, thus, the primary target for influenza vaccines. HA is a homotrimer consisting of head and stalk domains6. The head is the immunodominant domain and the primary target of antibodies generated by seasonal influenza vaccines or infection7,8,9,10. However, it is highly variable, making head-specific antibodies less effective against constantly evolving influenza virus variants. The HA stalk or stem is the immune-subdominant domain but is relatively more conserved11. Anti-stalk antibodies have been shown to correlate with protection in humans12,13. Furthermore, vaccines based on the conserved stalk domain have been reported to have a broader breadth14,15,16 and are considered to be an important target for the development of a universal or broadly protective influenza vaccine. Sequential vaccinations with heterologous chimeric hemagglutinins (cHAs) have enhanced stalk-specific antibody responses in small animal models and humans14,17,18. This strategy relies on the idea that sequential immunization with antigens having the same HA stalk, but very distant head domains, will boost the stalk-specific antibody responses by preferential recall of the memory B cells (MBCs) specific to the subdominant stalk domain as compared with naive responses to the immunodominant head domains.
Besides antigenic evolution, the waning of immunity poses a persistent challenge for current influenza vaccines. Seasonal influenza vaccine effectiveness may decline within a season, limiting its protective impact on vaccine recipients19,20. Resident long-lived bone marrow plasma cells (BMPCs) are important for maintaining the long-term antibody responses21 and, thus, protection from infection. Adjuvants have the potential to enhance the humoral immune response to vaccines and assist in the generation of antigen-specific BMPCs22,23, but the durability of those responses is still being investigated. There are various kinds of adjuvants available24. AS03 (GSK) is an adjuvant system containing DL-α-tocopherol and squalene in an oil-in-water emulsion. It has received regulatory approval for use in human vaccines, including Arepanrix and Pandemrix (GSK). In multiple studies, AS03 has been shown to improve antibody responses against influenza and coronavirus disease 2019 vaccines and boost the naive and cross-reactive MBC responses25,26,27,28,29,30,31. Incorporation of adjuvants into influenza vaccination strategies may potentially improve long-term immunity and address the challenges of waning vaccine effectiveness.
The nonhuman primate (NHP) immune system is more closely related to humans compared to other models like mice and ferrets. The expression of various Toll-like receptors on different cell types in NHPs is similar to their expression in humans, and this is an important consideration for studying the properties of adjuvants and their mechanisms of action32,33,34. In addition, NHPs are a good model to study the durability of immune responses induced by vaccines because they live much longer than mice, and serial tissue sampling can be done in the same animal. Thus, NHPs are an important model for studying the vaccine-induced immune response to human antigens and adjuvants.
In the current study, we characterized the magnitude, breadth and persistence of the antibody response elicited by an AS03-adjuvanted chimeric HA-based influenza vaccine, which was designed to induce robust anti-stalk-specific humoral immunity, in rhesus macaques. Humans are exposed to influenza virus antigens through either vaccination or infection. To mimic this, we first exposed rhesus macaques to influenza virus antigens by administering two doses of the seasonal quadrivalent influenza vaccine (QIV) and characterized the immune responses generated by this vaccine. Then, the rhesus macaques were primed with the cH8/1N1 (H8 head and H1 stalk) split vaccine and boosted with cH5/1N1 (H5 head and H1 stalk) split vaccine, mimicking the approach used in a human study17. We evaluated the impact of the AS03 adjuvant on humoral immunity and assessed the protective efficacy of the serum antibodies generated by the chimeric HA-based vaccine through challenge studies in passively immunized mice with heterologous influenza A viruses. Additionally, we monitored antibody and BMPC responses over a period of one year. Subsequently, the NHPs were immunized with a second dose of cH5/1N1, and we evaluated the presence and persistence of long-lived plasma cells (LLPCs) over nearly 2 years.
Results
Two doses of QIV generate poor HA stalk-specific antibody responses and no detectable BMPCs
To generate preexisting influenza virus-specific immunity and to understand the immune response induced by seasonal influenza vaccination in NHPs, we administered two doses of the seasonal inactivated QIV 6 weeks apart to a group of rhesus macaques (Fig. 1a). Blood was drawn from the immunized animals on days 0, 4, 7 and 14 to longitudinally monitor the plasmablast responses. We measured QIV-specific IgM, IgG and IgA antibody-secreting cells (ASCs) using an enzyme-linked immunospot (ELISpot) assay (Fig. 1b). QIV vaccinations induced low but detectable levels of QIV-specific IgM (Fig. 1c) and IgG (Fig. 1d) but not IgA (Fig. 1e) antibodies in the blood. The IgM responses were detectable on days 4–7 after prime and day 4 after boost. The IgG responses were detectable on day 7 after prime and day 4 after boost. These responses were transient and declined by day 14 after prime and day 7 after the boost. The magnitude of the IgM response was comparable after prime (geometric mean = 8 ASCs per million peripheral blood mononuclear cells (PBMCs)) and boost (geometric mean = 11 ASCs per million PBMCs); however, the magnitude of the IgG response was 6-fold higher after the boost (geometric mean = 62 ASCs per million PBMCs) compared to prime (geometric mean = 9 ASCs per million PBMCs). We also looked at H1 HA stalk-specific responses using chimeric HA antigens (cH5/1, cH6/1 and cH8/1) with stalks derived from the A/California/04/09 (pandemic H1N1) influenza A virus HA and heads of HAs not present in the QIV vaccine antigens17 (illustrations depicting the structural composition of the immunogens and proteins used in the study are shown in Extended Data Fig. 1). H1 HA stalk-specific IgG ASCs were mostly undetectable (Extended Data Fig. 2).
a, Study design. Ten rhesus macaques were given two doses of QIV at weeks 0 and 6. Blood was drawn on day 4 (D4) after immunization for plasmablast quantification. Blood and bone marrow aspirates were collected at different time points for serum antibody titer and ASC quantifications. b, Representative ELISpot for total plasmablasts (total ASCs) secreting IgG, IgM and IgA, and QIV-specific IgG, IgM and IgA after QIV booster dose on days 0, 4 and 7. Each well had 3.3 × 105 PBMCs. c–e, Longitudinal QIV-specific IgM (day 7 after prime, P = 0.0002; day 4 after boost, P = 0.0026) (c), IgG (day 4 after boost, P < 0.0001) (d) and IgA (e) plasmablast responses after QIV prime and boost represented as ASCs per million PBMCs. Statistical analysis was performed using Tukey’s multiple-comparisons test. All comparisons were made relative to the day of the first QIV immunization. f–h, Longitudinal serum IgG antibody responses against QIV (f), cH6/1 (g) and mini-HA (h) after QIV immunizations. Statistical analysis was performed using Tukey’s multiple-comparisons test. All comparisons were made relative to the day of the first QIV immunization. i, Spearman correlation, two-tailed, between serum antibody titers and plasmablasts. j, Representative ELISpot image for total IgG, QIV, cH8/1, cH6/1 and cH5/1 HA antigen-specific bone marrow plasma after two doses of QIV immunization. Bone marrow mononuclear cells (BMMCs; 3.3 × 105) were added to the top row and diluted 3-fold. k, Longitudinal QIV-specific BMPC response against QIV after QIV immunizations. The number of animals in this part of the study was n = 10 for all the assays. The dashed lines indicate the LoD. Two-way analysis of variance (ANOVA) was performed for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Panel a created with BioRender.com.
Next, we examined the QIV-specific serum IgG antibody responses (Fig. 1f). After prime, the titer was very low but there was a 6.5-fold boost at 2 weeks after the booster dose (geometric mean = 1,826) compared to the baseline (geometric mean titer = 279). We also looked at the stalk-specific antibody responses using cH6/1 and mini-HA. The change in the stalk-specific serum antibody titers was less than 2-fold (geometric mean titer = 338 for cH6/1, 274 for mini-HA) as compared to the baseline (geometric mean titer = 253 for cH6/1, 259 for mini-HA; Fig. 1g,h). We observed a positive correlation between the QIV-specific serum antibody titers on week 10 and IgG-secreting plasmablasts on day 4 after boost (Fig. 1i). We also monitored the QIV-specific LLPCs in the bone marrow by ELISpot assay. We could not detect any BMPCs for either QIV or stalk antigens (Fig. 1j,k). Overall, these data demonstrate that two QIV immunizations induce binding antibodies to the immunogen in serum but fail to induce stalk-specific antibody response and plasma cells in bone marrow.
AS03-adjuvanted chimeric HA vaccine enhances the magnitude of humoral immunity and induces LLPCs in bone marrow
After the two doses of QIV, the NHPs were divided into two groups and were immunized with 15 μg of chimeric HA vaccines, cH8/1N1 on week 21 and cH5/1N1 on week 33 (Fig. 2a). One group received AS03-adjuvanted vaccines (n = 7), and the other group received unadjuvanted vaccines (n = 8). We determined the antigen-specific plasmablast responses by ELISpot assay (Fig. 2b). We observed peak response on day 4 after cH5/1N1 immunization for both groups, but the response was significantly higher in the AS03-adjuvanted animals as compared to the unadjuvanted ones. We could detect ASCs in the blood of some of the animals in the adjuvanted group until day 14, but ASCs declined to undetectable levels in the unadjuvanted group by day 7 (Fig. 2c,d).
a, Schematic representation of study design. b, Representative ELISpot image comparing plasmablast responses between the unadjuvanted and AS03-adjuvanted groups on days 0, 4 and 8 after cH5/1N1 immunization. Each well had 3.3 × 105 PBMCs except day 4 for the AS03-adjuvanted group (1.1 × 105 PBMCs). c,d, Longitudinal comparison of cH8/1-specific (day 4, P = 0.0002; day 7/8, P = 0.0002) (c) and cH5/1-specific (day 4, P = 0.0003; day 7/8, P = 0.0003; day 14/15, P = 0.007) (d) IgG-secreting plasmablast responses after cH5/1N1 immunization between the AS03-adjuvanted (n = 7) and unadjuvanted (n = 8) groups. The dashed lines indicate the LoD. e,f, Longitudinal comparison of cH8/1-specific serum IgG titers at weeks 0, 6, 21, 23 (P = 0.0003), 27 (P = 0.0006), 33 (P = 0.0289), 35 (P = 0.0003), 41 (P = 0.0003), 53 (P = 0.0003), 65 (P = 0.0003), 77 (P = 0.0003) and 94 (P = 0.0003) (e), and cH5/1-specific IgG titers at weeks 0, 2, 6, 21, 23 (P = 0.0003), 27 (P = 0.0012), 35 (P = 0.0003), 41 (P = 0.0003), 53 (P = 0.0003), 65 (P = 0.0003), 77 (P = 0.0003) and 94 (P = 0.0003) (f), between the unadjuvanted (n = 8) and AS03-adjuvanted (n = 7) groups up to week 94. The dashed lines indicate the LoD. The error bars represent the geometric mean with geometric standard deviation. g,h, Comparison of MBC responses against cH8/1 at weeks 27 (P = 0.0039), 41 (P = 0.0039) and 65 (P = 0.0014) (g) and cH5/1 at weeks 27 (P = 0.0362), 41 (P = 0.0003) and 65 (P = 0.0002) (h) longitudinally between the AS03-adjuvanted (n = 7) and unadjuvanted (n = 8) groups. The dashed lines indicate the LoD. i, Representative ELISpot image for total, QIV, cH8/1, cH6/1 and cH5/1 HA antigen-specific BMPCs between unadjuvanted and AS03-adjuvanted groups at week 94. j,k, Longitudinal comparison of cH8/1-specific BMPC responses at week 25 (P = 0.0263), week 33 (P = 0.0014), week 37 (P = 0.0002), week 41 (P = 0.0002), week 53 (P = 0.0002), week 65 (P = 0.0002), week 77 (P = 0.0002) and week 94 (P = 0.0002) (j) and cH5/1-specific BMPC responses at week 33 (P = 0.0256), week 37 (P = 0.0002), week 41 (P = 0.0003), week 53 (P = 0.0002), week 65 (P = 0.0002), week 77 (P = 0.0002) and week 94 (P = 0.0002) (k) up to week 94 (n = 7 for adjuvanted, n = 8 for unadjuvanted groups). The dashed lines indicate the LoD. Pink represents unadjuvanted and blue represents AS03-adjuvanted animals. Fine lines represent individual animal values, whereas thick lines with symbols represent the geometric means. Two-tailed, unpaired Mann–Whitney U-test was performed for statistical significance between the unadjuvanted and AS03-adjuvanted groups for c–h, j and k. *P < 0.05, **P < 0.01, ***P < 0.001. The error bars for ELISA represent the geometric mean with geometric standard deviation. The black asterisk represents the comparison between the two groups. Panel a created with BioRender.com.
Serum antibody titers were tracked longitudinally for 94 weeks after the first QIV immunization. At 2 weeks after cH8/1N1 immunization, there was a 28-fold boost in cH8/1-specific IgG titer in the adjuvanted group (geometric mean = 7,453) as compared to a 3.7-fold change in the unadjuvanted group (geometric mean = 954; Fig. 2e). Similarly, at 2 weeks following cH5/1N1 immunization, there was a much greater change in the cH5/1-specific titers (9.5-fold) in the adjuvanted group (geometric mean = 10,765) as compared to 2.3-fold in the unadjuvanted group (geometric mean = 1,830; Fig. 2f). Following the peak titer, the antibody response showed a biphasic decay in both groups with a significant decline (3–7-fold from the peak) in the first 18 weeks followed by minimal changes in the next 40 weeks. The half-life estimates using the power law model revealed the t1/2 to be about 45–62 days for the first 18 weeks, and this was comparable between the two groups (Fig. 2e,f). By 20 weeks after boost, the cH8/1-specific and cH5/1-specific antibody titer reached baseline levels in the unadjuvanted group, whereas they were maintained at 6.5-fold higher levels through week 94 in the AS03-adjuvanted group. These results demonstrated that AS03-adjuvanted antibody persists for up to 15 months after the final immunization in serum.
To understand the immune mechanisms that contributed to the persistence of serum antibody response in the adjuvanted group, we determined the frequency of MBCs in the blood and LLPCs in the bone marrow. We determined the MBC frequencies at 6–8 weeks after each chimeric HA immunization (Fig. 2g,h). The AS03-adjuvanted animals showed strong induction of cH8/1-specific (0.5% at peak) and cH5/1-specific (0.9% at peak) MBCs following cH8/1 and cH5/1 immunizations and persisted through week 65 (8 months after boost). However, the cHA-specific MBCs were generally below our detection limit (except in two animals) in the unadjuvanted group. Similarly, the AS03-adjuvanted animals showed strong induction of LLPCs in bone marrow as early as 4 weeks after the cH8/1N1 immunization and peaked at 4 weeks (0.9% cH8/1; 1.0% cH5/1) after cH5/1N1 immunization (Fig. 2j,k). The cH5/1-specific LLPCs were low following cH8/1N1 immunization but were boosted following cH5/1N1 immunization. Impressively, the cHA-specific LLPCs persisted up to 94 weeks (15 months after the cH5/1N1 immunization) in the adjuvanted group and showed strong correlation with the serum antibody responses (Extended Data Fig. 3). As with the MBCs, the bone marrow LLPCs were generally below our detection limit in the unadjuvanted group. Overall, these results demonstrate that AS03 adjuvant helps to induce strong MBCs in blood and LLPCs in bone marrow, which associates with the persistence of serum antibody for more than one year.
AS03 adjuvant leads to higher vaccine-specific germinal center B cells in the lymph nodes
Adjuvants have been shown to enhance germinal center responses35,36. So, to assess the impact of AS03 on the germinal center B cells, fine needle aspirations from lymph nodes were taken at weeks 1 and 2, and a lymph node biopsy was performed at −2 and 3 weeks after cH5/1N1 immunization. Germinal center B cells were gated on live CD3−CD20+Ki-67+CD38− cells (Fig. 3a and Extended Data Fig. 4). We could not observe any significant difference in the frequencies of total germinal center B cells between the two groups. We used a cH5/1 receptor-binding site mutated probe, which is less sticky, to detect the cH5/1-specific germinal center B cells. We observed a significant increase in the cH5/1 probe-specific germinal center B cells (CD3−CD20+Ki-67+CD38− cH5/1-positive B cells) in the AS03-adjuvanted animals at week 3 after cH5/1 as compared to the unadjuvanted animals (Fig. 3b).
a, Representative flow plot showing the gating strategy for germinal center B cells and the frequency of germinal center B cells. Two-way ANOVA was performed for multiple comparisons. In the cH5/1 + AS03 group, significant differences were observed between week –2 and week 3 (P = 0.0153), and between week 1 and week 3 (P = 0.0452). b, Representative flow plot showing the gating strategy for cH5/1 probe-specific germinal center B cells and the corresponding frequencies. Two-way ANOVA was used for multiple comparisons. A significant difference was observed between the cH5/1 and cH5/1 + AS03 groups at week 3 (P = 0.0309). The number of animals was n = 8 in the unadjuvanted group and n = 7 in the adjuvanted group. Scatter dot plots display the mean, with error bars representing the standard deviation. The black asterisk represents the comparison between the unadjuvanted and AS03-adjuvanted groups at that time point. The blue asterisks represent significance within the adjuvanted group between the two time points. GC, germinal center. NS, not significant.
AS03-adjuvanted vaccine alters the innate immune cell composition, transcriptome and cytokine levels in the blood
AS03 is known to enhance the magnitude and breadth of the humoral immune responses to vaccine antigens in mice and humans by transiently modulating innate immune responses37. AS03 adjuvant has been shown to induce changes in chromatin accessibility in myeloid cells38. Therefore, we assessed changes in the composition of innate cells in the blood on days 0, 1, 2, 4, 7 and 14 after cH8/1N1 vaccinations (Fig. 4a,b) by flow cytometry analysis. We compared the changes in frequencies with baseline (day 0), and we observed a significant increase in the frequencies of non-classical (CD14−CD16+) and intermediate (CD14+CD16+) monocytes on days 1, 2 and 4, and a decrease in classical (CD14+CD16−) monocytes on days 1, 4, 7 and 14 after cH8/1N1 immunization in the adjuvanted group. Frequencies of monocytes also changed in the unadjuvanted group at certain time points. However, the differences were not significant between the two groups (Fig. 4b). We also observed changes in dendritic cell subsets in the blood on various days after vaccination in both groups compared to day 0, but there were no significant differences between the two groups at any time point (Extended Data Fig. 5).
a, Gating strategy for the monocyte and dendritic cell subsets. b, Frequencies of monocyte subsets (CD16+, non-classical; CD14+CD16+, intermediate; CD14+, classical) as a percentage of total lymphocytes. Data represent responses measured over 14 days following cH8/1 immunization. For non-classical monocytes, significant differences were observed in the unadjuvanted group on day 2 (P = 0.0242), and in the adjuvanted group on day 1 (P = 0.0017), day 2 (P = 0.0113) and day 4 (P = 0.0058) as compared to day 0. For intermediate monocytes, significant differences were observed in the unadjuvanted group on day 1 (P = 0.0418), day 7 (P = 0.0346) and day 14 (P = 0.0127); and in the adjuvanted group on day 1 (P = 0.0012), day 2 (P = 0.0412) and day 4 (P = 0.0098) as compared to day 0. For classical monocytes, significant differences were observed in the unadjuvanted group on day 7 (P = 0.0315) and day 14 (P = 0.0399), and in the adjuvanted group on day 1 (P = 0.0418), day 4 (P = 0.0385), day 7 (P = 0.0012) and day 14 (P = 0.0017) as compared to day 0. The number of animals was n = 8 in the unadjuvanted group and n = 7 in the adjuvanted group. Error bars represent the mean and s.e.m. A parametric t-test was used for statistical analysis. Blue and pink asterisks represent significance within that group. c, Graph showing the number of differentially expressed genes (DEGs) on days 1 and 2 in the AS03-adjuvanted and unadjuvanted animals as compared to day 0 after cH8/1N1 immunization. Blue represents AS03-adjuvanted, pink represents unadjuvanted, and orange represents the genes expressed significantly different between the two groups. d, Gene-set enrichment analysis pathway enrichment showing the significantly different gene sets between the two groups on day 1, and their over-expression or under-expression as compared to day 0 after cH8/1N1 immunization. NOM P val., nominal P value. e, Heat map depicts the log2-fold change of differentially expressed genes involved in interferon alpha response and the IL-6–JAK2–STAT3 pathway. Genes included with a log2-fold change of more than 1 and an adjusted P value < 0.05 are the first 50 leading edge genes from the gene-set enrichment analysis that contribute the most to the enrichment of the gene set on day 1 compared to day 0. f–i, Concentration (pg ml−1) of IL-6 (f), IL-5 (g), MIP-1α (h) and IL-1RA (i) in the plasma. For cytokine quantification, the number of animals on day 0 for both groups was n = 4. On days 1 and 2, the unadjuvanted group had n = 8, and the adjuvanted group had n = 7. Boxes represent the interquartile range (25th to 75th percentile), with the line indicating the median. Whiskers show minimum to maximum and all the points. Mixed-effects analysis was performed for multiple comparisons. The black asterisk represents the comparison between the unadjuvanted and AS03-adjuvanted groups at that time point. Blue and pink asterisks represent significance within that group between the two time points. For monocytes, a parametric t-test was performed. Blue and pink asterisks represent significance within that group as compared to day 0. The error bars represent the mean with s.e.m. The whiskers in the box-and-whisker plots represent all the points from minimum to maximum. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
We also examined changes in vaccine-induced gene expression on days 1 and 2 compared to pre-vaccination levels (day 0) after cH8/1N1 vaccination (Fig. 4c). In total, 547 and 59 genes were upregulated, and 302 and 28 were downregulated in the adjuvanted group on days 1 and 2, respectively. However, in the unadjuvanted group, 49 and 182 genes were upregulated, and 22 and 163 genes were downregulated on days 1 and 2, respectively (Fig. 4c). A total of 124 genes exhibited a significant difference in expression in the AS03-adjuvanted group as compared to the unadjuvanted groups on day 1. The expression of these genes was still elevated at day 2 but no longer significantly different from the unadjuvanted group. Most of these genes were involved in inducing a strong immune response, particularly interferon-stimulated genes such as GBP3, GBP7, IFIT2, IFIT3, IRF7, MX1, IDO1, OAS2 and OASL (Extended Data Fig. 6a). Gene-set enrichment analysis revealed significant enrichment of multiple pathways, including pathways related to inflammation and interferon (IFN) responses on days 1 and 2 between the two groups (Fig. 4d and Extended Data Fig. 6b). Additionally, genes involved in metabolic pathways showed considerable expression differences. Further dissection of the pathways showed that many genes involved in IFNα (Fig. 4e), IFNγ and complement pathways (Extended Data Fig. 6c,d) were enriched in the adjuvanted animals. There was also an upregulation in genes linked to the interleukin (IL)-6–JAK–STAT3 signaling pathway, including IRF1, MYD88, TYK2, STAT1, STAT2 and STAT3 (Fig. 4e). Interestingly, we noted upregulation of pathways related to platelet activation, megakaryocyte development and thrombopoietin, which have recently been shown to be associated with the induction of long-lived humoral immunity in humans39.
Next, we quantified the changes in the cytokine and chemokine levels in the plasma on days 1 and 2 after cH8/1N1 immunization using a Meso Scale Discovery (MSD) platform (Extended Data Fig. 7). We observed a significant increase in the cytokines IL-6, IL-5, MIP-1α and IL-1RA in the AS03-adjuvanted animals (Fig. 4f–i) as compared to the unadjuvanted group on day 1. Collectively, these data demonstrate that the AS03 adjuvant induces a strong pro-inflammatory response that has been shown to be associated with induction of a strong antibody response.
AS03 adjuvant improves the induction of influenza HA stalk-specific responses after chimeric HA vaccination
The objective of the chimeric HA vaccine strategy was to boost the HA stalk-specific responses. To test this, we used cH6/1 protein as used in the human trial17. This antigen features a head domain of H6 HA but includes the same stalk as present in the cH8/1 and cH5/1 immunogens. These monkeys are naive for H6 HA so a major response detected by this protein should be toward the stalk and potentially some toward cross-reactive head epitopes.
Following cH8/1N1 immunization, the cH6/1-specific plasmablasts were at the limit of detection (LoD) in some animals (Fig. 5a). However, the cH5/1N1 boost markedly increased their frequency, primarily in the AS03-adjuvanted animals. At day 4 after cH5/1N1 boost, the number of cH6/1-specific plasmablasts was 24-fold higher in the adjuvanted group (geometric mean = 149) compared to the unadjuvanted group (geometric mean = 6). The number of chimeric HA-specific plasmablasts specific for cH6/1, cH5/1 and cH8/1 was comparable in each group, suggesting that most of the plasmablasts induced after cH5/1N1 vaccination were likely stalk specific (Fig. 5b). Consistent with the plasmablast response, the adjuvanted animals showed a strong cH6/1-specific (Fig. 5c) and mini-HA-specific (Extended Data Fig. 8a) IgG response in serum following cH5/1N1 boost. At the peak (2 weeks after cH5/1 boost), the titer of cH6/1-specific IgG was 8-fold higher in the adjuvanted group (geometric mean = 8,739) compared to unadjuvanted group (geometric mean = 974). The higher cH6/1-specific IgG response in the adjuvanted group persisted at least until week 94 (about 15 months after boost). These data demonstrate that chimeric vaccination with AS03 adjuvant induces strong stalk-specific antibodies that persist for more than one year.
a, Longitudinal stalk-specific (cH6/1-specific) plasmablast responses were measured following chimeric HA immunizations, with significant increases observed at day 7 after cH8/1 immunization (P = 0.0476), day 4 after cH5/1 immunization (P = 0.0002) and day 7/8 after cH5/1 immunization (P = 0.0002). The number of animals on day 4 after cH8/1 immunization was n = 4 for both groups; at all other time points, the unadjuvanted group had n = 8 and the AS03-adjuvanted group had n = 7. b, Comparison of plasmablast responses to the chimeric antigens (cH8/1, cH5/1 and cH6/1) on day 4 after cH5/1N1 immunization (n = 7 for adjuvanted, n = 8 for unadjuvanted groups). Dunn’s multiple-comparison test was performed for statistical analysis. Boxes represent the interquartile range (25th to 75th percentiles), with the line indicating the median. Whiskers show minimum to maximum values and all the points. c, Longitudinal cH6/1-specific serum IgG antibody responses showed significant differences between the AS03-adjuvanted (n = 7) and unadjuvanted (n = 8) groups at multiple time points: week 23 (P = 0.0022), week 27 (P= 0.0059), week 33 (P = 0.0289), week 35 (P = 0.0003), week 41 (P = 0.0003), week 53 (P = 0.0003), week 65 (P = 0.0003), week 77 (P = 0.0003) and week 94 (P = 0.0003). The dashed lines indicate the LoD. The error bars represent the geometric mean with geometric standard deviation. d, Summary of polyclonal epitope mapping of serum antibodies at weeks 23, 33 and 35 against H1 hemagglutinin A/California/04/2009 (stabilizing mutation p.Glu47Lys in HA2). HA in the top left indicates the location of the three major stem epitopes. Three representative EMPEM maps derived from EMPEM analysis of animal 6 at week 35 are shown in the top right (angled stem in yellow with Protein Data Bank (PDB) ID 7T3D; low stem in green with PDB 5JW4; central stem in blue with PDB 4FQY). The bottom chart summarizes the epitopes targeted by polyclonal antibodies by animal and time point where a dot represents the existence of an epitope specific polyclonal antibody response (see Extended Data Fig. 9 for EMPEM data). NA, neuraminidase; RBS, receptor binding site. e, Longitudinal comparison of MBC responses against cH6/1 at week 27 (P = 0.0443), week 41 (P = 0.0003) and week 65 (P = 0.0002) between the AS03-adjuvanted (n = 7) and unadjuvanted (n = 8) groups. The dotted lines indicate the LoD. f, Longitudinal comparison of cH6/1-specific BMPC responses at weeks 21, 25, 33 (P = 0.007), 37 (P = 0.0002), 41 (P = 0.0002), 53 (P = 0.0002), 65 (P = 0.0002), 77 (P = 0.0002) and 94 (P = 0.0002) between the AS03-adjuvanted (n = 7) and unadjuvanted (n = 8) groups. The dotted lines indicate the LoD. g, Spearman correlation between the cH6/1 serum antibody titer and BMPCs at week 94. Black indicates correlation for all the animals. Blue indicates correlation for only AS03-adjuvanted animals. The dotted lines indicate the LoD. Pink represents unadjuvanted and blue represents AS03-adjuvanted animals. Fine lines represent individual animal values, whereas thick lines with symbols represent the geometric means. Two-tailed unpaired Mann–Whitney U-test was performed for statistical significance between the unadjuvanted and AS03-adjuvanted groups. The error bars for ELISA represent the geometric mean with geometric standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. The black asterisk represents the comparison between the unadjuvanted and AS03-adjuvanted groups at that time point.
To determine which epitopes were being targeted by the chimeric HA vaccination, we used electron microscopy polyclonal epitope mapping (EMPEM; Fig. 5d and Extended Data Fig. 8b). These results revealed in both groups that the dominant epitopes being targeted were indeed to the stalk region with a more diverse array of epitopes targeted in the adjuvanted group compared to the unadjuvanted group. For the unadjuvanted group, only a central stalk response, similar to human antibody CR9114 (ref. 40), was detected toward the stalk region and this only occurred in the final time point for animal 2. Animal 1 saw a single receptor-binding site region response in the final time point. Animals in the adjuvanted group all produced a central stalk response as well as a low stalk response that is similar in the angle of approach to the human antibody MEDI8852. MEDI8852, a human monoclonal antibody, binds to a highly conserved HA stalk epitope in the lower stalk and neutralizes a wide range of influenza A viruses41,42,43. Additionally, animal 5 produced an anchor response in the final time point, similar to human anchor antibody response 222-1C06 (ref. 44). Finally, animal 6 produced a response, described here as an angled stalk response, that utilizes an angle of approach not observed previously in human vaccine responses but is also distinct from the human anchor response (Fig. 5d). Overall, EMPEM analyses indicate that cHA vaccination in rhesus macaques elicits stalk-targeting polyclonal antibody responses, with more diverse stalk epitopes targeted in the AS03-adjuvanted group.
Next, we determined the HA stalk-specific long-term immune responses by evaluating MBCs in blood and BMPCs. Before chimeric HA vaccinations, there were no stalk-specific MBCs. After cH8/1N1 vaccination, there were some stalk-specific MBCs (geometric mean = 0.08% of total MBCs) in the adjuvanted animals, and they increased by 8-fold (geometric mean = 0.71%) after the cH5/1N1 vaccination. The frequency of MBCs persisted up to 32 weeks in the adjuvanted group (Fig. 5e). The stalk-specific MBCs were generally below our detection limit in the unadjuvanted animals. The adjuvanted animals also induced strong stalk-specific BMPCs at 4 weeks following cH5/1N1 immunization (0.70%). These responses showed an approximately 3-fold decline over the next 8 weeks, became more stable over time, and persisted for over a year (Fig. 5f). The cH6/1-specific BMPCs were undetectable in unadjuvanted animals. The serum antibody titers at week 94 correlated well with the BMPCs at that time (Fig. 5g).
These data suggest that addition of AS03 to the chimeric HA vaccination regimen helped in inducing strong, diverse and persistent HA stalk-specific humoral responses.
Passive transfer of serum from adjuvanted rhesus macaques protects mice against challenge with a lethal dose of heterologous strains of influenza virus
To determine the protective efficacy of vaccine-induced antibodies, we initially performed hemagglutination inhibition (HAI) assays to measure antibodies that block the binding of influenza virus HA to red blood cells. Our results showed that serum antibodies did not exhibit strong HAI activity against cH8/1Cal09N1Cal09, cH5/1Cal09N1Cal09, A/Guangdong-Maonan/SWL1536/2019 (H1N1) pdm09-like, B/Phuket/3073/2013 (B/Yamagata lineage)-like and B/Washington/02/2019 (B/Victoria lineage)-like viruses (Extended Data Fig. 9a–e). Next, we conducted microneutralization assays to evaluate serum antibodies that prevent infection of Madin–Darby canine kidney (MDCK) cells by the cH6/1N5 virus (Fig. 6a). We found that neutralizing antibody titers were nearly 10-fold higher in the adjuvanted group compared to pre-chimeric HA vaccines, whereas they were less than 2-fold higher in the unadjuvanted group at week 35. These data show that the stalk-specific antibodies are capable of neutralizing heterologous influenza A virus strains.
a, Comparison of microneutralization titers against cH6/1N5 influenza virus between the unadjuvanted and AS03-adjuvanted groups longitudinally with significant differences at weeks 35 (P = 0.0037) and 77 (P = 0.0433). The number of animals was n = 8 in the unadjuvanted group and n = 7 in the adjuvanted group. Mixed-effects analysis was performed for multiple comparisons. The error bars represent the mean with standard deviation. *P < 0.05, **P < 0.01. The black asterisk represents the comparison between the unadjuvanted and AS03-adjuvanted groups at that time point. b, Schematic diagram for passive antibody transfer and influenza virus challenge of mice. c,d, Weight loss (c) and survival (d) curves of mice passively immunized with serum from week 12 (8 weeks after QIV boost) and week 35 (2 weeks after cH5/1N1 immunization and peak stalk-specific serum IgG antibody titer) challenged with cH6/1N5 influenza virus. e,f, Weight loss (e) and survival (f) curves of mice passively immunized with serum from week 12 (8 weeks after QIV boost) and week 35 (2 weeks after cH5/1N1 immunization and peak stalk-specific serum IgG antibody titer) challenged with A/Netherlands/602/2009 or cH6/1N5 influenza viruses. The number of mice was n = 5 for each group. Survival curves were compared by log-rank (Mantel–Cox) test. Gray represents week 12 serum samples, pink represents unadjuvanted animals, and blue represents AS03-adjuvanted animals. Panel b created with BioRender.com.
First, we determined the mouse median lethal dose (mLD50) using serum collected 8 weeks after QIV booster (week 12). To test the protective efficacy of HA stalk-specific serum antibodies, serum of the rhesus macaques from different time points such as naive, week 12 and week 35 (2 weeks after cH5/1N1 vaccination with peak HA stalk-specific response) were pooled separately. Then, we passively transferred serum from immunized macaques into BALB/c mice and challenged them with a lethal dose (10 mLD50) of heterologous influenza virus strains cH6/1N5 (heterologous head and NA) and A/Netherlands/602/2009 virus (Fig. 6b). Following the challenge, all mice that received naive serum (Extended Data Fig. 9f,g) and serum from week 12 lost more than 25% of their body weight and succumbed to death by day 8. Similarly, peak vaccine serum (week 35) from unadjuvanted mice also failed to protect 4/5 mice from both viruses. However, peak vaccine serum from AS03-adjuvanted animals showed complete protection against both viruses (Fig. 6c,d), suggesting that stalk-specific antibodies are protective against a heterologous influenza A virus challenge.
AS03 leads to persistence of LLPCs in bone marrow and lymph nodes
In the human study17,18, the individuals were primed with cH8/1N1 and boosted with cH5/1N1. In the current study, we vaccinated rhesus macaques with a second dose of cH5/1N1 at week 94 (61 weeks after the first cH5/1N1 immunization), to investigate how antibody responses are affected when the animals are reexposed to the same HA. We monitored animals until weeks 193–206 (time of necropsy) to determine the persistence of LLPCs in bone marrow (Fig. 7a). We measured plasmablast responses specific to cH5/1 and cH6/1 on days 0, 4, 7 and 35 following cH5/1 boost and observed a 44–57-fold (44-fold for cH6/1 and 57-fold for cH5/1) higher plasmablast numbers in the adjuvanted group compared to the unadjuvanted group on day 4 (Fig. 7b,c). The magnitude of cH5/1-specific and cH6/1-specific plasmablasts within the adjuvanted group was comparable, suggesting that a major fraction of the response was directed against the stalk. Next, we assessed the serum antibody responses to the chimeric HA antigens cH5/1 (Extended Data Fig. 10a) and cH6/1 (Fig. 7d). Consistent with the plasmablast response, at day 14 (week 96), the serum antibody responses were 5–8-fold (5-fold for cH6/1 and 8-fold for cH5/1) higher in the AS03-adjuvanted group compared to the unadjuvanted group. These responses contracted 8–13-fold (8-fold for cH6/1 and 13-fold for cH5/1) in the AS03-adjuvanted group and 4–5-fold (4-fold for cH5/1 and 5-fold for cH6/1) in the unadjuvanted group over 100 weeks. We also compared the peak antibody responses between the first (week 35) and second (week 96) cH5/1N1 immunizations specific to cH5/1 and cH6/1 antigens to understand if there was a preferential boost toward H5 head and H1 stalk. The responses were comparable between the two time points in the adjuvanted group, but the unadjuvanted group exhibited a significant increase against both proteins (Fig. 7e). These data demonstrate that the second cH5/1 boost did not substantially alter the response toward the head and stalk portions of the chimeric HA.
a, Schematic representation of study design. b,c, Longitudinal comparison of cH5/1-specific (day 4, P = 0.0003; day 7, P = 0.0002; day 35, P = 0.007) (b) and cH6/1-specific (day 4, P = 0.0002; day 7, P = 0.0002) (c) IgG-secreting plasmablast responses after second cH5/1N1 immunization between the unadjuvanted (n = 8) and AS03-adjuvanted (n = 7) groups. The dashed lines indicate the LoD. d, Comparison of cH6/1-specific serum IgG titers at weeks 94 (P = 0.0003), 96 (P = 0.0003), 100 (P = 0.0012), 155 (P = 0.0003) and 193–206 (P = 0.0059) between unadjuvanted (n = 8) and AS03-adjuvanted (n = 7) groups. The dashed lines indicate the LoD. The error bars represent the geometric mean with geometric standard deviation. e, Comparison of peak antibody titers after 1st and 2nd cH5/1N1 immunizations against the cH5/1 and cH6/1 proteins for the AS03-adjuvanted (n = 7) and unadjuvanted (n = 8) animals. Boxes represent the interquartile range (25th to 75th percentiles), with the line indicating the median. Whiskers show minimum to maximum values and all the points. A paired t-test was performed for statistical significance for the change in the peak serum antibody titers in the animals. f, Comparison of cH6/1-specific BMPC responses at weeks 94 (P = 0.0002), 107 (P = 0.0002), 161 (P = 0.0002) and 193–206 (P = 0.0002) between the unadjuvanted (n = 8) and AS03-adjuvanted (n = 7) groups. g, Spearman correlation between cH6/1-specific IgG serum antibody versus BMPC frequencies at weeks 193–206. h, Affinity maturation of cH6/1 HA-specific IgG. The avidity index represents the percentage of cH6/1-specific antibody (measured by ELISA) that remains bound after washing with 8 M urea. Each data point corresponds to the mean of two assays. There were significant differences in the avidity indices at week 77 (P = 0.0142) and week 100 (P = 0.0094) and at the time of necropsy (P < 0.0001) between the AS03-adjuvanted (n = 7) and unadjuvanted (n = 8) groups. Scatterplot shows the lines at the mean with s.e.m. Statistical significance between groups and time points was determined using a two-way ANOVA. i, Representative ELISpot image for total IgG, cH5/1-specifc and cH6/1-specific IgG-secreting LLPCs in lymph nodes (LNs) at week 206. j, Frequencies of cH6/1-specific IgG-secreting plasma cells in the draining lymph nodes (LAxLN) (P = 0.007), blood, spleen and liver. Two-tailed unpaired Mann–Whitney U-test was performed for statistical significance between the unadjuvanted and AS03-adjuvanted groups for all the panels except e and h. The dashed lines indicate the LoD. The number of animals was n = 8 in the unadjuvanted group and n = 7 in the adjuvanted group for all the assays, unless otherwise indicated. Fine lines represent individual animal values, whereas thick lines with symbols represent the geometric means. The error bars for ELISA represent the geometric mean with geometric standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The black asterisk represents the comparison between the unadjuvanted and AS03-adjuvanted groups. Panel a created with BioRender.com.
We next monitored HA-specific plasma cells in the bone marrow over a 99–105-week period (weeks 193–206 after the first QIV immunization) following the second cH5/1N1 dose. On the day of the second cH5/1N1 boost, LLPCs were detected in the bone marrow of all animals in the adjuvanted group but not in the unadjuvanted animals (Fig. 7f and Extended Data Fig. 10b). Following the boost, we measured responses at weeks 13, 67 and 99–112, and these responses were maintained fairly constantly in 5 of 7 animals. At the time of necropsy, the frequency of cH6/1-specific bone marrow LLPCs showed a strong direct correlation with the cH6/1-specific serum antibody response (Fig. 7g).
We assessed changes in antibody affinity following heterologous boosts by performing 8 M urea affinity ELISA assays using cH6/1 HA to evaluate the extent of affinity maturation in stalk-specific antibodies. Our results revealed an increase in the avidity index of cH6/1-specific antibodies after each vaccination in the AS03-adjuvanted group, indicating affinity maturation due to the booster shots. In contrast, no such increase in avidity was observed in the unadjuvanted group, with the adjuvanted group showing higher avidity starting from week 77 compared to the unadjuvanted group (Fig. 7h). These findings highlight that the AS03 adjuvant enhances the avidity of the vaccine-induced antibody response.
We also examined the presence of plasma cells in the blood, draining lymph nodes, liver and spleen at necropsy. Impressively, HA-specific plasma cells were detected in the lymph nodes of five of seven animals (Fig. 7i,j and Extended Data Fig. 10c). These data demonstrate that AS03-adjuvant-induced plasma cells persist up to 2 years in bone marrow and draining lymph nodes.
Discussion
To advance the development of universal influenza vaccines, we tested the ability of the chimeric HA approach to induce a strong stalk-specific antibody response and to study how the AS03 adjuvant induces long-lived humoral immunity in NHPs. Like prior rodent and human studies15,17,18, our results demonstrated that this vaccine approach induces a strong stalk-specific antibody response providing protection against heterologous influenza virus infections. Importantly, our results demonstrated that the AS03 adjuvant generated LLPCs in the bone marrow and draining lymph nodes, and MBCs in blood, and this was associated with a durable antibody response. Furthermore, our results revealed the immunological mechanisms that potentially contributed to the induction of LLPCs. Collectively, these results establish the benefit of chimeric HA vaccination combined with the use of the AS03 adjuvant toward the development of a universal influenza virus vaccine.
We observed that after two doses of QIV, the stalk-specific ASCs were mostly undetectable, and there was less than a 2-fold change in the stalk-specific antibody responses in the serum in our NHP study. The seasonal influenza vaccines primarily induce antibodies targeting the head domain, and it is well-established that the HA head domain dominates the immune response, as it contains immunodominant antigenic sites that are the primary targets of neutralizing antibodies9,45. Recent studies have shown that chimeric HA vaccines are highly effective in eliciting stalk-specific immune responses in both mice and humans15,17,18. Our NHP study produced similar findings, with chimeric HA vaccine generating strong stalk-specific antibody responses, mirroring the results of human trials. However, there was a difference in the kinetics of induction of stalk-specific antibody response between humans and NHPs. In humans, peak stalk-specific serum antibody responses were observed immediately after the cH8/1N1 vaccination (first vaccination), and these titers were not significantly boosted following the cH5/1N1 vaccination (second vaccination). In the NHP study, peak stalk-specific responses were observed after the cH5/1N1 boost (second vaccination). We think this could be attributed to the presence of stalk-specific MBC responses in humans, which may have been generated by previous influenza virus infections or vaccinations. Also, the second cH5/1N1 boost did not significantly alter the immune response toward the head and stalk regions of the chimeric HA. This suggests that once a robust stalk-specific response, particularly in MBCs, has been established, further boosting of stalk-specific response may become more efficient.
A key goal of vaccination is to establish durable immunity, and the induction of bone marrow LLPCs is critical to achieve durability. Our study showed that two doses of QIV fail to generate detectable levels of QIV-specific BMPCs in NHPs. In fact, the BMPCs did not come up even after three additional cHA vaccinations in the unadjuvanted group. These data suggest that the current unadjuvanted QIV likely induces a poor BMPC response in humans. We recently reported the evaluation of BMPCs in humans following unadjuvanted influenza vaccination and showed that vaccination induced a small (2-fold) increase in BMPCs, but they declined within a year after vaccination46. We saw multiple differences between the studies in NHPs and humans. (1) In humans, we enriched the bone marrow mononuclear cells for plasma cells, whereas we did not do this in NHPs due to technical limitations such as lack of specific reagents to purify plasma cells. (2) Humans had nearly 1% influenza virus-specific BMPCs before the seasonal vaccination, whereas in the NHPs, the percentage was below our LoD (0.01%). (3) Humans have a complex exposure history of both influenza virus infections and vaccinations, and we are uncertain whether prior infections or vaccinations generated these LLPCs in the bone marrow. Earlier studies have demonstrated that viral infections induce long-lived BMPCs, which persist for extended periods21,47,48, whereas responses to nonreplicating protein antigens decline rapidly48.
A critical finding of our study is that AS03 adjuvant can help to induce durable influenza vaccine-specific BMPC response in NHPs, with persistence observed for up to 2 years (and potentially longer). This persistent BMPC response was associated with persisting levels of serum antibody response, which stabilized after an initial decline with no further waning during this phase. Studies in mice and NHPs comparing the effects of various adjuvants on severe acute respiratory disease coronavirus 2 (SARS-CoV-2) vaccines demonstrated that AS03 can induce BMPC responses49,50, but the durability of these BMPCs for the influenza vaccine has not been tested. In this study, we report the ability of AS03 to induce long-lasting BMPCs for up to 2 years. Our study also demonstrates that AS03 induces ASCs that persist for up to 2 years in draining lymph nodes. Activated B cells differentiate into ASCs soon after vaccination, and generally their presence in secondary lymphoid organs and blood is transient, typically lasting only a few days51,52. However, previous studies have demonstrated that SARS-CoV-2 mRNA-based vaccines can induce germinal center responses that persist for extended durations53,54,55. In our study, we did not measure germinal center responses after final vaccination, so we cannot determine whether the AS03-adjuvanted vaccine elicits similarly long-lived germinal center activity. Additionally, it remains to be investigated whether the ASCs found in the lymph nodes following AS03 vaccination are generated from ongoing, prolonged germinal center responses, and these ASCs subsequently migrate to the bone marrow to sustain the LLPC pool. We also do not know how the presence of these LLPCs in the draining lymph nodes will impact the response to future booster vaccinations. Overall, these findings suggest that incorporating adjuvants like AS03 into protein-based influenza vaccines may play a crucial role in establishing and sustaining long-term vaccine-induced immunity.
Next, we attempted to understand the mechanisms driving the enhanced immune responses observed with the AS03 adjuvant in NHPs, as the molecular basis of its adjuvanticity remains less well-defined56,57,58. Earlier studies in mice and humans37,59,60 observed changes in the composition of monocytes and dendritic cells after administration of the AS03-adjuvanted vaccine. In NHPs, we also observed changes in monocyte composition, along with some dendritic cell subsets, in the blood 1–2 days after vaccination; however, the differences were subtle between the adjuvanted and unadjuvanted groups. Our RNA-sequencing analysis revealed that many of the genes involved in immune response were upregulated early after vaccination. Many of these upregulated genes like IFITM2, GBP1, IFIT1, IFIT2, IFIT3, MX1, OAS2 and OASL were also reported earlier in AS03-adjuvanted H1N1 vaccine studies in humans59,60. Furthermore, gene-set enrichment analysis highlighted the upregulation of pathways associated with the IL-6–JAK–STAT3 signaling cascade. In our panel of cytokines, we observed an increase in IL-5 and IL-6, corroborating previous studies49,59,61. IL-5, originally introduced as a T cell-replacing factor, leads to the differentiation of activated B cells into ASCs and to enhanced antibody levels62,63. IL-6 plays a vital role in B cell proliferation, maturation and the survival of plasma cells, contributing to elevated IgG secretion64,65,66. Importantly, IL-6 has been identified as a key factor in differentiating human MBCs into LLPCs in vitro, promoting immunoglobulin production and sustaining their presence for at least 4 months67. Thus, the enhanced IL-6 production induced by the AS03 adjuvant might be one of the reasons for the generation and persistence of LLPCs in the bone marrow. A recent study defined the molecular and immunological mechanisms that contribute to the generation of long-lived humoral immunity following influenza vaccination with and without AS03 in humans and showed an important role for platelets in inducing durable humoral immunity via TPO-mediated megakaryocyte activation39. Interestingly, we also noted that AS03 adjuvant induces pathways related to platelet activation, megakaryocyte development and TPO in our NHP study. Our NHP data corroborate the findings in humans for AS03 adjuvant.
In our NHP study, animals were rendered preimmune to influenza by prior seasonal vaccinations, and not by natural infection. This is different from what is observed in humans where both seasonal vaccinations and natural infections likely contribute to the generation of immunity to influenza. Natural influenza virus infection is known to elicit stronger stalk-specific antibodies than inactivated vaccines, both in animal models and humans68,69. For example, children with prior H1N1 infection exhibited detectable levels of H1 stalk-reactive antibodies, while these antibodies were largely absent in influenza-naive children69. We think that the induction of stalk-specific antibodies and MBCs by natural infection before cHA vaccination might induce more potent stalk-specific antibodies after the first cHA (cH8/1) vaccination. Indeed, in our human study, we observed a substantial boost in stalk-specific antibody response just after the cH8/1 vaccination with AS03 (refs. 17,18). These findings support the idea that an infected immune background may lead to a more rapid or potent stalk-specific immune response following AS03-adjuvanted cHA vaccination, and future studies should explore how different exposure histories affect long-term immunity. Finally, it is important to consider how this cHA vaccine approach would perform in pediatric or immunologically naive populations. Based on preclinical and human studies, we would expect that two doses of cHA + AS03 in infants or young children would effectively prime strong stalk-specific antibody responses.
Another important consideration is how prior immunity induced by the adjuvanted seasonal influenza vaccines may influence the immune landscape following cHA vaccination. MF59-adjuvanted vaccine has been shown to enhance antibody avidity, increase HA stalk-specific titers and extend the duration of immunity70. These findings suggest that seasonal vaccination with adjuvanted formulations may provide a priming effect, enhancing or modulating the cHA vaccine-induced response. Further investigation is needed to understand how pre-exposure to adjuvanted vaccines impacts the efficacy of AS03-adjuvanted cHA vaccines.
In summary, our study shows that the failure of the seasonal influenza vaccine to induce a long-lasting antibody response is associated with its inability to induce BMPCs, and highlights the contribution of AS03 adjuvant in inducing BMPCs and lymph node ASCs that persist for up to 2 years. It also reveals the transcriptomic signatures of AS03 adjuvant that could be important for the induction of these long-lived BMPCs in NHPs. These results have important implications for developing vaccines that induce durable immunity.
Methods
Animal subjects and ethical approval
Male Indian rhesus macaques around 3 to 5 years of age from the Emory National Primate Research Center breeding colony were obtained in accordance with the Animal Welfare Act and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals using protocols approved by Emory University Institutional Animal Care and Use Committee.
All mouse experiments and procedures were approved by the Institutional Animal Care and Use Committee of the University of Wisconsin–Madison School of Veterinary Medicine (protocol no. V006426-A04). Six- to eight-week-old female BALB/c mice were used for the study. Animals were acclimated to the facility conditions (25–28 °C and 35–45% humidity) before the start of experiments, allowed access to food and water (ad libitum), kept on a 12/12- h light–dark cycle, and provided with enrichment. Humane endpoints for euthanasia included body weight loss greater than 25% or inability to remain upright.
Study design
Fifteen rhesus macaques were initially immunized with two full doses of Fluzone quadrivalent influenza virus vaccine (Fluzone Quadrivalent 2020–2021, 0.5 ml single-dose, pre-filled syringe, UT7011KA, Sanofi Pasteur) into the deltoid muscle with a gap of 4 weeks between the doses. Subsequently, animals were divided into two groups and immunized with cHA vaccines. The split virion-based chimeric HA vaccines were kindly provided by GSK. More details about the chimeric HA vaccines are available in the previously published article17. Full human doses (15 µg) of the vaccines in a volume of 0.5 ml with either phosphate buffered saline (PBS) or AS03 (GSK) were administered intramuscularly. The first immunization administered was cH8/1N1 followed by cH5/1N1, at week 33. An additional cH5/1N1 dose was given with or without AS03 adjuvants in week 94. Additionally, ten more monkeys were immunized with two full doses of the same QIV, spaced 6 weeks apart, to evaluate stalk-specific responses in the blood and bone marrow.
ELISA
The ELISA was performed for binding antibody response to seasonal QIV, cH8/1, cH5/1, cH6/1 and mini-H1 following the previously described methods with some modifications71. In brief, Costar high-binding microtiter plates (Corning Life Sciences) were coated overnight at 4 °C with 1 µg ml−1 of the antigens in PBS. The following day, the plates were washed with 0.05% PBS with Tween-20 (PBST), followed by blocking for 30 min at room temperature (RT) with 5% whey diluted in the PBST. Rhesus macaque serum was serially diluted threefold, and incubated for 1 h at RT. After washing the plates again with PBST, the bound serum antibodies were detected using a 1:10,000 dilution of anti-rhesus IgG (Accurate Chemical and Scientific) conjugated to horseradish peroxidase (HRP). The plates were washed with PBST again, and then tetramethylbenzidine substrate (KPL) was added. After 20 min, the reaction was stopped by adding 100 µl of 2 N H2SO4. A wavelength of 450 nm was used to read the plates in a Varioskan Lux (Thermo Fisher Scientific) reader. Prism software was used to calculate 50% maximal binding (EC₅₀) values by using the log(inhibitor) versus normalized response (variable slope) method, with 0.1 as the cutoff value.
ELISpot assay
Assays were performed according to previously established protocols with some modifications23. Capture antibody was prepared at a concentration of 2 µg ml−1 in 1× PBS for IgG and IgA (Rockland Immunochemicals Affinity Purified Anti-Monkey 617-101-130), and for IgM (BD Pharmingen Purified Mouse Anti-Human IgM 555780) and the QIV, cH5/1, cH6/1 and cH8/1 antigens were diluted to 1 µg ml−1 in PBS. Each well of the ELISpot plates (MSHAN4B50, Millipore-Sigma) was coated with 100 µl of the antigen solution, and was incubated overnight at 4 °C. The next day, plates were washed once with wash buffer I (1× PBS, 0.05% Tween-20) followed by blocking with 100 µl of complete Roswell Park Memorial Institute (RPMI) medium, containing 10% fetal bovine serum (FBS) from Corning (35-011-CV) and a 1:100 dilution of penicillin G and streptomycin (10,000 U penicillin per ml, 10,000 µg streptomycin per ml) from Lonza (17-602E), and incubated for 2 h at 37 °C in a 5% CO2 environment. PBMCs or bone marrow mononuclear cells were resuspended to a concentration of 107 cells per ml. A volume of 50 µl (approximately 5 × 105 cells per well) was added to the first well of each column, followed by 3-fold serial dilutions in the subsequent wells. After an incubation of 16–18 h, the plates were washed 4× with both wash buffers I and II. Next, the plates were incubated with 100 µl of biotin-conjugated anti-monkey IgG antibody (1:2,000 dilution; 617-106-012, Rockland), IgM (1:2,000 dilution; 617-106-007, Rockland) and IgA (1:2,000 dilution; 109-065-011, Jackson ImmunoResearch) for 2 h at RT. Afterward, the plates were washed 4× with wash buffer I. Then, solution containing 1% FBS and HRP avidin D (1:5,000 dilution; A-2004, Vector Laboratories) was added to the wells for 2 h at RT. Following another 4× washes with wash buffer II and then with buffer I, the plates were developed using 3-amino-9-ethylcarbazole (AEC; 0.3 mg ml−1 AEC in 0.1 M sodium acetate buffer, pH 5.0, with 0.03% hydrogen peroxide). Once dried, the plates were analyzed with the Immunospot CTL counter and Image Acquisition 4.5 software (Cellular Technology) until week 94. Plates after week 94 were read by a Mabtech IRIS fluorospot/ELISpot reader. Spots were counted manually, and ASCs were reported as either per million PBMCs or as a percentage of total BMPCs.
MBC ELISpot assay
PBMCs were thawed and subsequently cultured in complete RPMI medium supplemented with R848 (2.5 mg ml−1; tlrl-r848, InvivoGen) and IL-2 (1,000 U ml; 200-02, PeproTech) at a concentration of 1 million cells per ml. On day 3, half of the complete medium containing IL-2 and R848 was replaced with fresh medium. On day 6, the cells were washed, counted and then resuspended in complete RPMI medium. Five hundred thousand cells were added to the ELISpot plates that had been coated with antigens, followed by 3-fold serial dilutions in the subsequent wells, and incubated for 8 h. The ELISpot assay was performed as described above.
HAI inhibition assay
Rhesus macaque serum samples were incubated with RDE derived from Vibrio cholerae (Denka Seiken) at 37 °C for 18–24 h. Sodium citrate (2.5%) was added subsequently, and the mixture was subjected to heat treatment at 55 °C for 60 min. The RDE-processed serum was subsequently adjusted to a 1:10 dilution using 1× PBS. In a V-bottom 96-well microplate, 50 µl of the treated serum was serially diluted 2-fold in 1× PBS up to a 1:2,560 dilution. Four hemagglutination units of the respective viral strains were introduced into each well, bringing the total volume to 50 µl, followed by incubation on a shaker at RT for 30 min. Next, 50 µl of 0.5% turkey red blood cells (Lampire Biological Laboratories) suspended in 1× PBS were added to the serum–virus mixture and incubated at 4 °C for 45–60 min. The HI titers were determined as the inverse of the highest serum dilution that completely inhibited hemagglutination of four units of the virus.
Microneutralization
The microneutralization assay was performed as described earlier72. The neutralization of influenza virus (cH5/1N1) was evaluated using MDCK cells. MDCK cells were seeded at a density of 20,000 cells per well in 96-well cell culture plates (Corning) and incubated overnight at 37 °C in a humidified incubator with 5% CO2. The following day, sera treated with receptor-destroying enzyme were diluted 2-fold across a 96-well plate in infection medium, which consisted of 1× minimum essential media (MEM; Gibco), 1.2% bovine serum albumin (BSA; MP Biomedicals), 2 mM L-glutamine (Gibco), 100 U ml−1 penicillin, 100 µg ml−1 streptomycin (Gibco), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Gibco), 3.2% NaHCO₃ (Sigma-Aldrich) and 1 µg ml−1 L-(tosylamido-2-phenyl)ethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich). Next, 60 µl of a 100× median tissue culture infectious dose (TCID₅₀) of virus in infection medium was mixed with 60 µl of the serially diluted sera in a new 96-well plate and incubated on a shaker for 1 h at RT. After incubation, 100 µl of the sera–virus mixture was added to the PBS-washed MDCK cells, which were then incubated for 1 h at 33 °C with 5% CO2. The virus inoculum was aspirated, and the MDCK cells were washed with 200 µl of PBS. One hundred microliters of the serially diluted sera was added to the corresponding wells and incubated for 72 h at 33 °C with 5% CO2. After the 72-h incubation, 50 µl of the cell culture supernatant was transferred to V-bottom 96-well plates (Thermo Fisher Scientific) along with 50 µl of 0.5% chicken red blood cells. The plates were incubated for 45 min at 4 °C, and the presence or absence of hemagglutination was assessed. The data were reported as the endpoint titer, representing the lowest dilution at which no hemagglutination was observed.
Germinal center B cell staining
Lymph node cells were stained with a cocktail of surface antibodies specific for CD3 (clone SP34-2; BD Biosciences), CD20 (clone 2H7; BD Biosciences), CD38 (clone OKT10, NHP Reagent Resources), LIVE/DEAD Near-IR Dead Cell stain (Life Technologies) and cH5/1 B cell probes conjugated to FITC and APC for 20 min in the dark at RT. Red blood cells were lysed using Lysing Solution (BD Biosciences) for 10 min in the dark. After lysis, cells were spun down at 2,000 rpm (Beckman Coulter Allegra X-12R centrifuge) for 5 min and washed with 2 ml of FACS wash buffer (PBS with 2% FBS and 0.05% sodium azide) and again spun down at 2,000 rpm for 5 min. FACS wash was decanted, and cells were fixed and permeabilized with BD FACS Permeabilizing Solution 2 (BD, Biosciences) according to the manufacturer’s protocol and stained with Ki-67 (clone B56; BD Biosciences) for 30 min in the dark at RT. Cells were again washed with FACS buffer and acquired on an LSR Fortessa (BD, Biosciences). Cells from frozen lymph nodes collected at week −4 (pre-vaccination) were used as naive controls for the cH5/1 probe staining.
Innate immune cell staining
Plasma was removed from whole blood by spinning at 2,000 rpm (Beckman Coulter Allegra X-12R centrifuge) for 10 min to prevent plasma antibodies from binding to the CD16 receptor and blocking anti-CD16 binding. Blood was reconstituted back to the original volume with PBS. Blood was then stained with LIVE/DEAD Near-IR Dead Cell stain and antibodies specific for CD3, CD20, CD14 (clone M5E2, BioLegend), CD16 (clone 3G8, BD Biosciences), HLA DR (clone G46-6, BD Biosciences), CD1c (clone L161, BioLegend), CD141 (clone 1A4, BD Biosciences), CD123 (clone 7G3, BD Biosciences), CD11c (clone 3.9, BioLegend), CD159 (clone NKG-2A, Miltenyi Biotec), CD69 (clone FN50, BD Biosciences), CD86 (clone IT2.2, BioLegend) and CD80 (clone L307.4, BD Biosciences) and incubated at RT for 20 min in the dark. Red blood cells were lysed similarly to the above germinal center B cell staining protocol, spun down and washed with FACS wash. From here, cells were acquired on an LSR Fortessa.
Mesoscale cytokine analysis
The MSD U-PLEX Viral Combo 1 (NHP) kit (K15344K-1) was used to measure cytokine concentration in plasma. The kit measures 19 analytes: G-CSF, GM-CSF, IFNα2a, IFNγ, IL-1RA, IL-1β, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IP-10, MCP-1, MIP-1α, tumor necrosis factor and VEGF-A. The MSD-provided protocol for U-PLEX assays was followed with no changes. Briefly, after bringing all reagents to RT, the two multiplex coating solutions were added to the appropriate 96-well plate (50 μl per well), and the plates were covered and incubated for 1 h while shaking at 600 rpm on a Lab-Line Instruments Titer plate shaker. After washing three times with 1× MSD wash buffer (R61AA-1), two replicates of serial 5-fold dilutions of calibrator mixes were added to the appropriate plates, along with two blank wells. Plasma was diluted 2-fold and added to appropriate wells (50 μl per well). Plates were incubated while shaking for 1 h and washed again, and then detection antibody master mix was added to the appropriate plates. After another 1-h incubation and wash, MSD GOLD Read Buffer B was added to each well and the plate was immediately read using the MESO QuickPlex SQ 120 MM instrument.
EMPEM
Digestion was quenched with iodoacetamide. Quenched digestion reactions were incubated with a small amount of Capture Select IgG-Fc resin to deplete free Fc and undigested IgG before concentration and size-exclusion chromatography. For each EMPEM dataset, 500 μg of polyclonal Fab (pFab) was incubated with 10 μg of the target HA (A/California/04/2009 E47K HA1/2) overnight at RT and then purified using size-exclusion chromatography to isolate pFab–antigen complexes. Grids were prepared for negative stain EMPEM by depositing 3 μl of purified Fab–antigen complexes at ~20 μg ml−1 onto glow-discharged, carbon-coated 400-mesh copper grids (Electron Microscopy Sciences). Samples were blotted and stained with 2% wt/vol uranyl formate using standard blotting procedures. Grids were imaged on either a Talos 200 C with a Falcon II direct electron detector and a CETA 4K camera (FEI) at 200 kV, ×73,000 magnification and 1.98 Å per pixel; a Tecnai Spirit T12 (FEI) with a CMOS 4K camera (TVIPS) at 120 kV, ×52,000 magnification and 2.06 Å per pixel; or a Tecnai T20 (FEI) with an Eagle CCD 4K camera (FEI) at 200 kV, ×62,000 magnification, and 1.67 Å per pixel. Micrographs were collected using Leginon, particles were picked and stacked using Appion, and processed using Relion73,74,75,76. UCSF Chimera was used to visualize and analyze models generated with Relion and to generate figures.
Transcriptomics
Blood samples were collected in PAXgene Blood RNA tubes (BD Biosciences), and RNA was extracted using the MagMAX for Stabilized Blood Tubes RNA Isolation Kit, designed for use with PAXgene Blood RNA Tubes (Thermo Fisher Scientific). The quality of the RNA was evaluated with a TapeStation 4200 (Agilent), after which 1 μg of total RNA underwent globin transcript depletion using the GLOBINclear Kit, human (Thermo Fisher Scientific). For cDNA synthesis, 10 ng of the globin-depleted RNA was used as input with the Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio), following the manufacturer’s protocol. The amplified cDNA was fragmented and dual-indexed barcodes were added using the Nextera XT DNA Library Preparation kit (Illumina). Libraries were validated through capillary electrophoresis on a TapeStation 4200 (Agilent), pooled in equal concentrations and sequenced with PE100 reads on an Illumina NovaSeq 6000, with approximately 40 million reads per sample.
Passive immunization
Serum samples from the animals of unadjuvanted and AS03-adjuvanted groups were pooled in equal volumes for each time point. Six- to eight-week-old female BALB/c mice received 200 µl of serum via intraperitoneal injection (n = 5 per group). Twenty-four hours later, before the viral challenge, small blood samples were collected through the submandibular route to confirm successful serum transfer (via ELISA). The mice were then challenged with 10 LD₅₀ of either the A/Netherlands/602/2009 virus or the cH6/1N5 virus (titered in animals that had passively received rhesus macaque serum 8 weeks after QIV booster immunization). Mice were monitored daily for survival and body weight changes over a 14-day period following infection. Mice experiencing more than 25% body weight loss were considered to have reached the experimental endpoint and were euthanized.
Affinity ELISA
High-binding ELISA plates (96-well flat-bottom immune plate MaxiSorp Cert, N/Ster, PS, 439454, Thermo Fisher Scientific) were coated with 100 µl per well of recombinant cH6/1 HA protein at a concentration of 1 µg ml−1 in PBS and incubated overnight at 4 °C. The following day, the plates were washed once with 1× PBST (1× PBS, 0.05% Tween-20) and then blocked with 100 µl per well of blocking buffer consisting of PBS supplemented with 0.05% Tween-20 and 10% FBS for 90 min at RT. After blocking, 50 µl of serum, diluted at a 1:33.3 ratio in blocking buffer, was added to the first well of each row, followed by serial 3-fold dilutions across the plate. The plates were incubated for 90 min at RT and subsequently washed four times with PBST (PBS + 0.05% Tween-20). Next, 100 µl per well of HRP-conjugated goat anti-rhesus IgG (GAMon/IgG(Fc)/PO, Nordic MUBio), diluted 1:10,000 in blocking buffer, was added and incubated for 90 min at RT. The plates were again washed four times with PBST, followed by an additional four washes with PBS to remove residual detergent. Colorimetric detection was performed by adding 100 µl per well of o-phenylenediamine dihydrochloride substrate (SIGMAFAST, peroxidase substrate, chromogenic, tablet P9187, Sigma-Aldrich). After color development for 16 min, the reaction was quenched by adding 100 µl per well of 1 M HCl, and absorbance was measured at 490 nm using a microplate reader.
Next, affinity ELISA was performed according to the protocol outlined above, with some modifications as described in the previously published method77. Briefly, plasma samples were diluted based on prior serial dilution ELISA results (described above) to achieve an optical density reading close to 1 at 490 nm, and 100 µl of the appropriate plasma dilution was added to each well. The plates were incubated for 90 min at RT. Subsequently, the plates were washed four times with PBST, followed by incubation with 200 µl per well of 8 M urea in PBS for 5 min at RT. Afterward, the plates were washed four times with PBST. Next, 100 µl per well of HRP-conjugated goat anti-rhesus was added followed by addition of o-phenylenediamine dihydrochloride substrate and stopping of the reaction by addition of 1 N HCl. The avidity index was calculated as the ratio of optical density on the urea-wash plate to the optical density on the PBS-wash plate, expressed as a percentage.
Statistical analysis
Statistical analysis was done using GraphPad Prism version 10.3.1. Two-way ANOVA was performed for multiple comparisons. The Mann–Whitney U-test was performed for the comparison between two groups at a given time point. A paired t-test was used for comparisons between two time points within a group. An unpaired parametric t-test was used for comparisons between two groups. The Spearman correlation was used for correlations between serum antibody titer and ASCs. P values < 0.05 were considered statistically significant.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
Electron microscopy maps were deposited to the Electron Microscopy Data Bank under accession codes EMD-49315, EMD-49316, EMD-49317, EMD-49318, EMD-49319, EMD-49320, EMD-49321, EMD-49322, EMD-49323 and EMD-49324. The RNA-sequencing data for this study have been deposited in the NCBI Gene Expression Omnibus under accession no. GSE294334. Source data are provided with this paper. Other data are available from the corresponding author upon reasonable request.
Code availability
The scripts used for the transcriptomics analysis are available at Zenodo via https://doi.org/10.5281/zenodo.17094472 (ref. 78).
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Acknowledgements
We sincerely thank V. Vassilev, B. Bouzya, N. Dendouga and L. Warter from GSK for providing split virion-based chimeric HA vaccines and AS03. Their support has been invaluable to our research. AS03, Arepanrix and Pandemrix are trademarks owned by or licensed to the GSK group of companies. Fluzone is a trademark of Sanofi Pasteur. This study was funded by the Collaborative Influenza Vaccine Innovation Centers (CIVICs) contract no. 75N93019C00051 to F.K., R.A., R.R.A., A.B.W. and Y.K.; UM1AI169662 (R.R.A), and Office of Research Infrastructure Programs (ORIP)/NIH base grant P51 OD011132 to ENPRC. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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Contributions
A. Akhtar contributed to study conceptualization, designed experiments, performed peripheral blood, BMPC and MBC assays, analyzed and interpreted the data and wrote the paper. T.M.S. contributed to study conceptualization, designed experiments, performed ELISA, flow cytometry for innate immune cells and germinal center B cells, analyzed and interpreted the data and wrote the paper. C.G., G.N., H.A. performed passive immunization experiments in mice under the supervision of Y.K. A.N.L., S.T.R., J.A.F. and J.H. performed the EMPEM under the supervision of A.B.W. K.S. performed mesoscale cytokine analysis under the supervision of R.R.A. S.S. produced chimeric HA antigens for the assays under the supervision of F.K. A. Abbad and J.M.C. performed HAI and microneutralization assays under the supervision of F.K. C.H. and H.A. conducted half-life analysis of serum antibodies under the supervision of R. Antia. G.K.T. performed transcriptomic analysis. U.M. helped with coordinating the study, sample processing and retrieving samples for affinity ELISA. C.W.D. contributed to study conceptualization and wrote the paper. F.K. and P.P. conceptualized the study, designed experiments and wrote the paper. R.R.A. and R. Ahmed conceptualized the study, designed experiments, provided overall supervision and wrote the paper. All authors contributed to paper editing.
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The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines influenza virus vaccines (including cHAs) and influenza virus therapeutics which list F.K. and P.P. as coinventors and F.K. has received royalty payments from some of these patents. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, Castlevax, to develop SARS-CoV-2 vaccines. F.K. is cofounder and scientific advisory board member of Castlevax. F.K. has consulted for Merck, GSK, Sanofi, Curevac, Seqirus and Pfizer and is currently consulting for Third Rock Ventures, Gritstone and Avimex. The laboratory of F.K. is also collaborating with Dynavax on influenza vaccine development and with VIR on influenza virus therapeutics. J.H. and A.B.W. are consultants for Third Rock Ventures. The laboratory of A.B.W. received unrelated sponsored research agreements from Third Rock Ventures during the conduct of the study. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Illustrations depicting the structural composition of QIV, chimeric HA, and mini-HA antigens.
(A) The quadrivalent influenza vaccine (QIV) contains hemagglutinin (HA) proteins from four different influenza virus strains. The chimeric HAs (B-D) used in this study consist of a conserved H1 stalk domain paired with variable head domains derived from other subtypes. The mini-HA (E) represents a headless HA construct, comprising only the stalk domain. Illustrations were made on power point.
Extended Data Fig. 2 Stalk-specific ASC responses post QIV immunizations.
(A) Representative HA stalk-specific ELISpot. Longitudinal stalk-specific (chimeric HA antigen-specific) IgG secreting ASC responses post QIV immunizations as enumerated by ELISpot assay against cH8/1 (B), cH5/1 (C), and cH6/1 (D) antigens. The number of animals in this part of the study was n = 10 for all the assays in this figure. The dotted lines indicate the limit of detection (LoD). Two-way ANOVA was performed for multiple comparisons.
Extended Data Fig. 3 Correlation between serum antibody titers and BMPCs.
Spearman correlation between the cH8/1 (A) and cH5/1 (B) serum antibody titer and BMPCs at week 94 (for AS03-adjuvanted, n = 7; unadjuvanted, n = 8). Number in black indicates correlation for all the animals and the number in blue indicates correlation for only AS03-adjuvanted animals.
Extended Data Fig. 4 GC B cell gating strategy.
Representative gating strategy for GC B cells and cH5/1 probe-specific GC B cells. Top panel represents staining of cells from lymph nodes from naïve animals. Bottom panel represents staining of cells from lymph nodes at week 36 (3 weeks post cH5/1 immunization).
Extended Data Fig. 5 Longitudinal analysis of DC subsets post cH8/1 immunization.
Frequencies of DC subsets (pDCs, CD11c, BDCA1 + , BDCA1 + BDCA3 + , BDCA3 + , BDCA1- BDCA3- as percent of total lymphocytes. Data represent responses measured over 14 days following cH8/1 immunization. For pDCs, significant differences were observed in the unadjuvanted group on Day 1 (p = 0.015) and Day 2 (p = 0.0178) as compared to Day 0. For BDCA1 + , significant differences were observed in the unadjuvanted group on Day 7 (p = 0.0313) and in the adjuvanted group on Day 1 (p = 0.0369), Day 2 (p = 0.0346), Day 4 (p = 0.0384) and Day 7 (p = 0.0309) as compared to Day 0. For BDCA1 + BDCA3 + , significant differences were observed in the unadjuvanted group on Day 1 (p = 0.0007), Day 2 (p = 0.0196), and Day 7 (p = 0.0006), and in the adjuvanted group on Day 1 (p = 0.0195), Day 2 (p = 0.0064), Day 4 (p = 0.0438) and Day 7 (p = 0.0004) as compared to Day 0. The number of animals was n = 8 in the unadjuvanted group and n = 7 in the adjuvanted group. For BDCA3 + , significant differences were observed in the unadjuvanted group on Day 1 (p = 0.0264), Day 2 (p = 0.0028), Day 4 (p = 0.0199) and Day 7 (p = 0.0417), and in the adjuvanted group on Day 4 (p = 0.0118) as compared to Day 0. For BDCA1-BDCA3-, significant differences were observed in the unadjuvanted group on Day 7 (p = 0.0239) and in the adjuvanted group on Day 1 (p = 0.0122), Day 2 (p = 0.0269), Day 4 (p = 0.0128) and Day 14 (p = 0.0324) as compared to Day 0. For AS03-adjuvanted, n = 7 and unadjuvanted, n = 8. Error bars represent the mean and SEM. A parametric t-test was used for statistical analysis. Blue and pink asterisk represent significance within that group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Extended Data Fig. 6 Gene expression and pathway analysis post-cH8/1 immunization.
(A) Top 50 genes (by descending fold change) out of 124 deferentially expressed genes which were significantly different (by adjusted p-value < 0.05) between the AS03 adjuvanted and unadjuvanted groups on day 1 post cH8/1 immunization. (B) Comparison of gene-set pathway enrichment analysis between the AS03-adjuvanted and unadjuvanted groups on days 1 and 2, and between days 1 or 2 vs day 0 within the groups. All the gene sets except platelet activation, factors involved in megakaryocyte development and TPO pathway are from hallmark pathway database. Platelet activation signaling and aggregation and factors involved in megakaryocyte development and platelet production gene sets are from Reactome, and TPO pathway gene sets are from Biocarta databases. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C & D) Heat map of gene-set involved in IFN-γ (C), and complement (D) pathway.
Extended Data Fig. 7 Plasma cytokine and chemokine analysis.
Measurement of 19 plasma cytokine and chemokine concentrations on days 0, 1, and 2 by Meso Scale Discovery (MSD) U-PLEX Viral Combo 1 (NHP) kit. For cytokine quantification, the number of animals on day 0 for both groups was n = 4. On days 1 and 2, the unadjuvanted group had n = 8, and the adjuvanted group had n = 7. Boxes represent the interquartile range (25th to 75th percentile), with the line indicating the median. Whiskers show minimum to maximum and all the points.
Extended Data Fig. 8 Stalk-specific serum antibody titers and their structural characterization.
(A) Longitudinal mini-HA-specific serum IgG antibody responses showed significant differences between the AS03-adjuvanted (n = 7) and unadjuvanted (n = 8) groups at multiple time points: Week 23 (p = 0.0093), Week 27 (p = 0.0093), Week 33 (p = 0.4634), Week 35 (p = 0.0003), Week 41 (p = 0.0006), Week 53 (p = 0.0003), Week 65 (p = 0.0401), Week 77 (p = 0.0003), and Week 94 (p = 0.0003). The dotted lines indicate the limit of detection (LoD). Error bars represent the geometric mean ± geometric standard deviation. Statistical significance between the unadjuvanted and AS03-adjuvanted groups was assessed using a two-tailed unpaired Mann–Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Black asterisk (*) represents comparison between the two groups. Negative stain EM reconstructions of pAbs in complex with recombinant A/California/04/2009 E47K HA2 at weeks 23, 33, and 35 (B). Animals 1 and 2 are unadjuvanted whereas animals 3-6 are AS03-adjuvanted. Stem specificity: red- RBS region, purple-anchor, yellow-angled stem, green-low stem, blue-central stem.
Extended Data Fig. 9 HAI titers against various strains and animal survival Following passive immunization.
HAI titer against cH8/1Cal09N1Cal09 (A), cH5/1Cal09N1Cal09 (B), A/Guangdong-Maonan/SWL1536/2019 (H1N1) pdm09-like virus (C), B/Phuket/3073/2013 (B/Yamagata lineage)-like virus (D), B/Washington/02/2019 (B/Victoria lineage)-like virus (E). cH5/1Cal09N1Cal09 and cH8/1Cal09N1Cal09 viruses HA head domain were derived respectively from the HA of A/Vietnam/1203/2004 (H5N1) virus and the HA of A/mallard/Sweden/24/02 (H8N4) virus, the stalk domain and NA of both viruses was derived from A/California/04/09 (H1N1) and the internal genes were derived from A/Puerto Rico/8/34 (H1N1) virus. The dotted lines indicate the limit of detection (LoD). Two-way ANOVA was performed for multiple comparisons. The number of samples was n = 8 in the unadjuvanted group and n = 7 in the adjuvanted group. Weight loss and survival curve of mice passively immunized with serum from naive serum challenged with A/Netherlands/602/2009 (F) and cH6/1N5 (G) influenza viruses. The number of mice was n = 5 for each group. Survival curves were compared by Log-rank (Mantel-Cox) test. Error bar for weight loss represent mean with SD.
Extended Data Fig. 10 cH5/1-Specific responses post second cH5/1 immunization.
(A) Longitudinal comparison of cH5/1-specific serum IgG titers at weeks 94 (p = 0.0003), 96 (p = 0.0003), 100 (p = 0.0012), 155 (p = 0.0003), 193-206 (p = 0.0006) between the unadjuvanted (n = 8) and AS03-adjuvanted (n = 7) groups up to Week 94. The error bars for represent the geometric mean with geometric standard deviation. (B) Comparison of cH6/1-specific BMPC responses post cH5/1N1 second dose at weeks 94 (p = 0.0002), 107 (p = 0.0003), 161 (p = 0.0002), 193-206 (p = 0.0002) between the AS03-adjuvanted (n = 7) and unadjuvanted (n = 8) groups. (C) Frequencies of cH5/1-specific IgG secreting plasma cells in the draining lymph nodes (LAxLN) (p = 0.007), blood, spleen, and liver. For AS03-adjuvanted, n = 7 and unadjuvanted, n = 8. Two-tailed Mann-Whitney U test was performed for statistical significance between the unadjuvanted and AS03 adjuvanted groups for all the assays in this figure. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Black asterisk (*) represents comparison between the two groups.
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Akhtar, A., Styles, T.M., Gu, C. et al. Influenza vaccine based on AS03-adjuvanted chimeric HA induces long-lived stalk-specific plasma cells in bone marrow and lymph nodes of nonhuman primates. Nat Immunol 26, 2045–2058 (2025). https://doi.org/10.1038/s41590-025-02309-1
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DOI: https://doi.org/10.1038/s41590-025-02309-1
