Comparison of adjuvants to optimize influenza neutralizing antibody responses

Seasonal influenza vaccines represent a positive intervention to limit the spread of the virus and protect public health. Yet continual influenza evolution and its ability to evade immunity pose a constant threat. For these reasons, vaccines with improved potency and breadth of protection remain an important need. We previously developed a next-generation influenza vaccine that displays the trimeric influenza hemagglutinin (HA) on a ferritin nanoparticle (NP) to optimize its presentation. Similar to other vaccines, HA-nanoparticle vaccine efficacy is increased by the inclusion of adjuvants during immunization. To identify the optimal adjuvants to enhance influenza immunity, we systematically analyzed TLR agonists for their ability to elicit immune responses. HA-NPs were compatible with nearly all adjuvants tested, including TLR2, TLR4, TLR7/8, and TLR9 agonists, squalene oil-in-water mixtures, and STING agonists. In addition, we chemically conjugated TLR7/8 and TLR9 ligands directly to the HA-ferritin nanoparticle. These TLR agonist-conjugated nanoparticles induced stronger antibody responses than nanoparticles alone, which allowed the use of a 5000-fold-lower dose of adjuvant than traditional admixtures. One candidate, the oil-in-water adjuvant AF03, was also tested in non-human primates and showed strong induction of neutralizing responses against both matched and heterologous H1N1 viruses. These data suggest that AF03, along with certain TLR agonists, enhance strong neutralizing antibody responses following influenza vaccination and may improve the breadth, potency, and ultimately vaccine protection in humans.     

Dissociation of TRIF bias and adjuvanticity

Toll-like receptors (TLRs), a family of “pattern recognition receptors,” bind microbial and host-derived molecules, leading to intracellular signaling and proinflammatory gene expression. TLR4 is unique in that ligand-mediated activation requires the co-receptor myeloid differentiation 2 (MD2) to initiate two signaling cascades: the MyD88-dependent pathway is initiated at the cell membrane, and elicits rapid MAP kinase and NF-κB activation, while the TIR-domain containing adaptor inducing interferon-β (TRIF)-dependent pathway is initiated from TLR4-containing endosomes and results in IRF3 activation. Previous studies associated inflammation with the MyD88 pathway and adjuvanticity with the TRIF pathway. Gram-negative lipopolysaccharide (LPS) is a potent TLR4 agonist, and structurally related molecules signal through TLR4 to differing extents. Herein, we compared monophosphoryl lipid A (sMPL) and E6020, two synthetic, non-toxic LPS lipid A analogs used as vaccine adjuvants, for their capacities to activate TLR4-mediated innate immune responses and to enhance antibody production. In mouse macrophages, high dose sMPL activates MyD88-dependent signaling equivalently to E6020, while E6020 exhibits significantly more activation of the TRIF pathway (a “TRIF bias”) than sMPL. Eritoran, a TLR4/MD2 antagonist, competitively inhibited sMPL more strongly than E6020. Despite these differences, sMPL and E6020 adjuvants enhanced antibody responses to comparable extents, with balanced immunoglobulin (Ig) isotypes in two immunization models. These data indicate that a TRIF bias is not necessarily predictive of superior adjuvanticity.     

A novel combination of intramuscular vaccine adjuvants,nanoemulsion and CpG produces an effective immune response against influenza A virus

BackgroundVaccination is the most effective approach to prevent infection with highly pathogenic avian influenza (HPAI). Adjuvants are often used to induce effective immune responses and overcome the immunological weakness of recombinant HPAI antigens. Given the logistical challenges of immunization to HPAI during pandemic situations, vaccines administered via the intramuscular (I.M.) route would be of value.MethodsA new formulation of nanoemulsion adjuvant (NE02) suitable for I.M. vaccination was developed. This NE02 was evaluated alone and in combination with CpG to develop H5 immune responses in mouse and ferret models. Measures of recombinant H5 (rH5) specific immunity evaluated included serum IgG and IgG subclasses, bronchoalveolar lavage fluid IgA, and cytokines. The activation of NF-kB was also analyzed. The efficacy of the vaccine was assessed by performing hemagglutination inhibition (HAI), virus neutralization (VN) assays, and viral challenges in ferrets.ResultsI.M. vaccination with rH5-NE02 significantly increased rH5-specific IgG and protected ferrets in the viral challenge model providing complete protection and sterile immunity in all animals tested. Combining NE02 and CpG produced accelerated antibody responses and this was accompanied by an elevation of IFN-γ and IL-17 responses and the downregulation of IL-5. The combination also caused a synergistic effect on NF-kB activation. In immunized ferrets after viral challenge, the rH5-NE02 + CpG vaccine via I.M. achieved at least 75% and 88% seroconversion of HAI and VN antibody responses, respectively, and improved body temperature stabilization and weight loss over NE02 alone.ConclusionsThe I.M. injection of NE02 adjuvanted rH5 elicits strong and broad immune responses against H5 antigens and effectively protects animals from lethal H5 challenge. Combining this adjuvant with CpG enhanced immune responses and provided improvements in outcomes to viral challenge in ferrets. The results suggest that combinations of adjuvants may be useful to enhance H5 immune responses and improve protection against influenza infection.     

Licensed Bacille Calmette-Guérin (BCG) formulations differ markedly in bacterial viability,RNA content and innate immune activation

BackgroundBacille Calmette-Guérin (BCG), the live attenuated tuberculosis vaccine, is manufactured under different conditions across the globe generating formulations that may differ in clinical efficacy. Innate immune recognition of live BCG contributes to immunogenicity suggesting that differences in BCG viability may contribute to divergent activity of licensed formulations.MethodsWe compared BCG-Denmark (DEN), -Japan (JPN), -India (IND), -Bulgaria (BUL) and -USA in vitro with respect to a) viability as measured by colony-forming units (CFU), mycobacterial membrane integrity, and RNA content, and b) cytokine/chemokine production in newborn cord and adult peripheral blood.ResultsUpon culture, relative growth was BCG-USA > JPN ≫ DEN > BUL = IND. BCG-IND and -BUL demonstrated >1000-fold lower growth than BCG-JPN in 7H9 medium and >10-fold lower growth in commercial Middlebrook 7H11 medium. BCG-IND demonstrated significantly decreased membrane integrity, lower RNA content, and weaker IFN-γ inducing activity in whole blood compared to other BCGs. BCG-induced whole blood cytokines differed significantly by age, vaccine formulation and concentration. BCG-induced cytokine production correlated with CFU, suggesting that mycobacterial viability may contribute to BCG-induced immune responses.ConclusionsLicensed BCG vaccines differ markedly in their content of viable mycobacteria possibly contributing to formulation-dependent activation of innate and adaptive immunity and distinct protective effects.     

Use of protective antigen of Bacillus anthracis as a model recombinant antigen to evaluate toll-like receptors 2, 3, 4, 7 and 9 agonists in mice using established functional antibody assays,antigen-specific antibody assays and cellular assays

Toll-like receptor (TLR) agonists can act as immune stimulants alone or as part of alum or oil formulations. Humoral and cellular immune responses were utilized to assess quantitative and qualitative immune response enhancement by TLR agonists using recombinant protective antigen (rPA) of B. anthracis as a model antigen. To rPA, combined with aluminum hydroxide (Alhydrogel; Al(OH)3) or squalene (AddaVax?), was added one of 7 TLR agonists: TLR2 agonist Pam3CysSK4 (PamS), TLR3 agonist double stranded polyinosinic:polycytidylic acid (PolyIC), TLR4 agonists Monophosphoryl lipid A (MPLA) or glucopyranosyl lipid A (GLA), TLR7-8 agonists 3M?052 or Resiquimod (Resiq), or TLR9 agonist CPG 7909 (CPG). CD-1 or BALB/c mice received two intraperitoneal or intramuscular immunizations 14 days apart, followed by serum or spleen sampling 14 days later. All TLR agonists except PamS induced high levels of B. anthracis lethal toxin-neutralizing antibodies and immunoglobulin G (IgG) anti-PA. Some responses were >100-fold higher than those without a TLR agonist, and IP delivery (0.5 mL) induced higher TLR-mediated antibody response increases compared to IM delivery (0.05 mL). TLR7-8 and TLR9 agonists induced profound shifts of IgG anti-PA response to IgG2a or IgG2b. Compared to the 14-day immunization schedule, use of a shortened immunization schedule of only 7 days between prime and boost found that TLR9 agonist CPG in a squalene formulation maintained higher interferon-γ-positive cells than TLR4 agonist GLA. Variability in antibody responses was lower in BALB/c mice than CD-1 mice but antibody responses were higher in CD-1 mice. Lower serum 50% effective concentration (EC50) values were found for rPA-agonist formulations and squalene formulations compared to Al(OH)3 formulations. Lower EC50 values also were associated with low frequency detection of linear peptide epitopes. In summary, TLR agonists elicited cellular immune responses and markedly boosted humoral responses.     

Systematic literature review of neutralizing antibody immune responses to non-vaccine targeted high-risk HPV types induced by the bivalent and the quadrivalent vaccines

IntroductionStudies on the cross-protective effect of HPV bivalent and quadrivalent vaccines demonstrated inconsistent findings against additional HPV types covered by the nonavalent vaccine. The objective of this study was to conduct a systematic literature review to assess the consistency and durability of the cross-protective neutralizing antibody immune responses of the currently licensed bivalent and quadrivalent vaccines to non-vaccine HPV types targeted by the nonavalent vaccine (HPV 6, 11, 31, 33, 45, 52, and 58).MethodsPubMed and EMBASE databases were searched from 2008 to 2019 to identify studies reporting antibody/immune response after vaccination with either the bivalent, quadrivalent, or nonavalent vaccine. Key outcomes were seroconversion, seropositivity or geometric mean titers against HPV types 6, 11, 31, 33, 45, 52, and 58.ResultsEighteen publications met inclusion criteria, reporting on 14 interventional and five observational studies. Across all studies, immune responses to non-vaccine high-risk HPV types after bivalent vaccination were higher than baseline or quadrivalent vaccine. Nonavalent vaccine elicited near total seroconversion to HPV types 31, 33, 45, 52, and 58, with seropositivity remaining near 100% up to 24 months post-dose 1. In contrast, bivalent and quadrivalent vaccination resulted in lower seroconversion levels for non-vaccine types, which waned over time.ConclusionsThe cross-protection antibody/immune response among participants having received all three doses of bivalent or quadrivalent vaccine is not comparable to the specific response elicited by HPV vaccine types. Even in cases where a statistically significant cross-reactive immunological response is reported, long-term data on the duration of the response beyond two years are very limited. Further, the lack of a standard for assays limits comparability of results between studies.     

Vaccination with HepB-CpG vaccine in individuals undergoing immune suppressive drug therapy

BackgroundImmunosuppressed patients are a targeted group for HBV vaccination but suboptimal antibody responses occur when traditional recombinant vaccines are used.MethodsWe tested an FDA approved immune adjuvanted HBV vaccine (HEPLISAV--B® or HepB-CpG) in medically immune suppressed individuals. HepB-CpG was given to 10 patients taking biologic agents or anti-rejection therapy. Each received vaccine at time 0 and week 4 with a third dose at week 12 if anti-HBs remained less than 10 mIU/mL.ResultsSeroprotective anti-HBs developed in 70 % of participants by week 24. Those taking biologic agents responded more rapidly and a third dose was generally needed in those transplanted. By week 24, most taking biologics but only 2 of 6 on anti-rejection treatment had antibody levels exceeding 100 mIU/mL.ConclusionsSeroprotective anti-HBs developed in 70 % with HepB-CpG. Antibody responses were more rapid in those taking biologic agents but a third dose improved antibody responses in transplanted participants.     

Increases in HPV-16/18 antibody avidity and HPV-specific memory B-cell response in mid-adult aged men post-dose three of the quadrivalent HPV vaccine

Strong quantitative and functional antibody responses to the quadrivalent human papillomavirus (HPV) vaccine were reported in mid-adult aged men, but there are limited data on the avidity of the antibody response and the memory B-cell response following vaccination. Although circulating antibodies induced by vaccination are believed to be the main mediators of protection against infection, evaluation of avidity of antibodies and memory B cell responses are critical for a better understanding of the vaccine immunogenicity mechanisms. Both the modified enzyme-linked immunosorbent assay (ELISA) and the enzyme-linked immunosorbent spot (ELISpot) assay are tools to measure the humoral and cellular immune responses post vaccination to characterize vaccine immunogenicity. The avidity of HPV-16 and HPV-18 specific IgG in the serum of mid-adult aged men (N = 126) who received three quadrivalent HPV vaccine doses was examined using a modified ELISA. HPV-16 memory B-cell responses were assessed via ELISpot at month 0 (prior to vaccination) and 1-month post-dose three of the vaccine (month 7). The quadrivalent vaccine induced an increase in HPV-16 and HPV-18 antibody avidity at month 7. HPV-18 avidity levels moderately correlated with anti-HPV-18 antibody titers, but no association was observed for HPV-16 antibody titers and avidity levels. The HPV-16-specific memory B-cell response was induced following three vaccine doses, however, no association with anti-HPV-16 antibody avidity was observed. Three doses of quadrivalent HPV vaccine increased antibody affinity maturation for HPV-16/18 and increased the frequency of anti-HPV-16 memory B-cells in mid-adult aged men.     

Safety and immunogenicity of Multimeric-001 (M-001) followed by seasonal quadrivalent inactivated influenza vaccine in young adults – A randomized clinical trial

BackgroundThe continuing evolution of influenza viruses poses a challenge to vaccine prevention, highlighting the need for a universal influenza vaccine. We evaluated the safety and immunogenicity of one such candidate, Multimeric-001 (M-001), when used as a priming vaccine prior to administration of quadrivalent inactivated influenza vaccine (IIV4).MethodsHealthy adults 18 to 49 years of age were enrolled in a phase 2 randomized, double-blind placebo-controlled trial. Participants received two doses of either 1.0-mg M-001 or saline placebo (60 per study arm) on Days 1 and 22 followed by a single dose of IIV4 on about Day 172. Safety, reactogenicity, cellular immune responses and influenza hemagglutination inhibition (HAI) and microneutralization (MN) were assessed.ResultsThe M-001 vaccine was safe and had an acceptable reactogenicity profile. Injection site tenderness (39% post-dose 1, 29% post-dose 2) was the most common reaction after M-001 administration. Polyfunctional CD4+ T cell responses (perforin-negative, CD107α-negative, TNF-α+, IFN-γ+, with or without IL-2) to the pool of M-001 peptides increased significantly from baseline to two weeks after the second dose of M-001, and this increase persisted through Day 172. However, there was no enhancement of HAI or MN antibody responses among M-001 recipients following IIV4 administration.ConclusionsM-001 administration induced a subset of polyfunctional CD4+ T cells that persisted through 6 months of follow-up, but it did not improve HAI or MN antibody responses to IIV4. (clinicaltrials.gov NCT03058692).     

A study to evaluate the immunogenicity and shedding of live attenuated influenza vaccine strains in children 24–<48 months of age

BackgroundQuadrivalent live attenuated influenza vaccine (LAIV4) showed reduced effectiveness against the A/H1N1 component in the 2013–2014 and 2015–2016 influenza seasons. The most likely cause of reduced LAIV effectiveness against A(H1N1)pdm09 strains was poor intranasal replication.ObjectivesTo compare the immunogenicity and shedding of a new A/H1N1 strain (A/Slovenia), to a A/H1N1 strain known to have reduced effectiveness (A/Bolivia).Patients/methodsThis was a randomized, double-blind, multicenter study. Children aged 24–<48 months of age were randomized 1:1:1 to receive two doses of LAIV4 2017–2018 (LAIV4_A/Slovenia), or LAIV4 2015–2016 or trivalent LAIV (LAIV3) 2015–2016 formulations (LAIV4_A/Bolivia or LAIV3_A/Bolivia, respectively) on days 1 and 28. The primary endpoint was strain-specific hemagglutination inhibition (HAI) antibody seroresponse at 28 days post each dose, and secondary endpoints included immunogenicity, shedding, and safety. Solicited symptoms, adverse events (AEs), and serious AEs (SAEs) were recorded. Pre-specified statistical testing was limited to the primary endpoint of HAI antibody responses.ResultsA total of 200 children were randomized (median age 35.3 months; 53% male; 57% had previously received influenza vaccine). Significantly higher HAI antibody responses for the A/Slovenia strain were observed after Dose 1 and Dose 2. Neutralizing antibodies and nasal immunoglobulin A antibody responses were higher for A/Slovenia versus A/Bolivia. More children shed the A/Slovenia vaccine strain than the A/Bolivia strain on Days 4–7 after Dose 1. No deaths, SAEs, or discontinuations from vaccine occurred.ConclusionsThe new A(H1N1)pdm09 A/Slovenia LAIV strain demonstrated improved immunogenicity compared with a previous strain with reduced effectiveness and induced immune responses comparable to a highly efficacious pre-pandemic H1N1 LAIV strain. These results support the use of LAIV4 containing A/Slovenia as a vaccine option in clinical practice.     

Antibody dependent disease enhancement (ADE) after COVID-19 vaccination and beta glucans as a safer strategy in management

A potential risk associated with vaccines for COVID-19 is antibody-dependent disease enhancement (ADE) in which vaccine induced antibody mediated immune responses may lead to enhanced SARS CoV- 2 acquisition or increased disease severity. Though ADE has not been clinically demonstrated with any of the COVID-19 vaccines so far, when neutralizing antibodies are suboptimal, the severity of COVID-19 has been reported to be greater. ADE is presumed to occur via abnormal macrophages induced by the vaccine based immune response by antibody-mediated virus uptake into Fc gamma receptor IIa (FcγRIIa) or by the formation of Fc-mediated excessive antibody effector functions. Beta-glucans which are naturally occurring polysaccharides known for unique immunomodulation by capability to interact with macrophages, eliciting a specific beneficial immune-response and enhancing all arms of the immune system, importantly without over-activation are suggested as safer nutritional supplement-based vaccine adjuvants for COVID-19.     

Astragalus polysaccharides combined with simvastatin as an immunostimulant enhances the immune adjuvanticity of oil-in-water emulsion and immune responses in mice

Oil-in-water emulsion-based adjuvants have demonstrated acceptable safety in many disease indications, while their adjuvant activities for vaccines still need to be improved. Recently, the strategy of combining adjuvants with multiple types of immunostimulants has been shown to enhance immune responses. In this study, astragalus polysaccharides were combined with simvastatin as an immunostimulant to construct a compound O/W emulsion adjuvant. The formulations were optimized according to the OVA-specific antibody responses induced in mice. For this reason, high (5 mg/mL), medium (2.5 mg/mL), and low (1.25 mg/mL) concentrations of astragalus polysaccharides and high (10 mg/mL), medium (1 mg/mL), and low (0.1 mg/mL) concentrations of simvastatin were selected. The final optimal formulation of the immunostimulant was a high concentration of astragalus polysaccharides combined with a medium concentration of simvastatin. The optimal compound O/W emulsion adjuvant could induce effective humoral and cellular immune responses that were stronger and more stable than those induced by aluminum adjuvant and Freund's adjuvant. The OVA/HAPS-MSim-OE induced dramatically strong and persistent IgG expressions and Th1-polarized immune responses. What’s more, the highest CD4⁺/CD8⁺lymphocyte ratios were observed in OVA/HAPS-MSim-OE group. In addition, compound O/W emulsion adjuvant groups significantly promoted the secretion of IFN-γ and IL-6, which also indicated that the compound O/W emulsion adjuvants could induce both enhanced Th1 and Th2-mediated immune responses but prefer the Th1-mediated ones. This study would contribute to an interesting and promising direction in the development of emulsion-based adjuvants.     

Single-dose combination nanovaccine induces both rapid and durable humoral immunity and toxin neutralizing antibody responses against Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax, continues to be a prominent biological warfare and bioterrorism threat. Vaccination is likely to remain the most effective and user-friendly public health measure to counter this threat in the foreseeable future. The commercially available AVA BioThrax vaccine has a number of shortcomings where improvement would lead to a more practical and effective vaccine for use in the case of an exposure event. Identification of more effective adjuvants and novel delivery platforms is necessary to improve not only the effectiveness of the anthrax vaccine, but also enhance its shelf stability and ease-of-use. Polyanhydride particles have proven to be an effective platform at adjuvanting the vaccine-associated adaptive immune response as well as enhancing stability of encapsulated antigens. Another class of adjuvants, the STING pathway-targeting cyclic dinucleotides, have proven to be uniquely effective at inducing a beneficial inflammatory response that leads to the rapid induction of high titer antibodies post-vaccination capable of providing protection against bacterial pathogens. In this work, we evaluate the individual contributions of cyclic di-GMP (CDG), polyanhydride nanoparticles, and a combination thereof towards inducing neutralizing antibody (nAb) against the secreted protective antigen (PA) from B. anthracis. Our results show that the combination nanovaccine elicited rapid, high titer, and neutralizing IgG anti-PA antibody following single dose immunization that persisted for at least 108 DPI.     

Phosphorothioated antisense oligodeoxynucleotide suppressing interleukin-10 is a safe and potent vaccine adjuvant

While vaccination is highly effective for the prevention of many infectious diseases, the number of adjuvants licensed for human use is currently very limited. The aim of this study was to evaluate the safety, efficacy, and to clarify the mechanism of a phosphorothioated interleukin (IL)-10-targeted antisense oligonucleotide (ASO) as an immune adjuvant in intradermal vaccination. The cytotoxicity of IL-10 ASO and its ability to promote T cell proliferation were assessed by Cell Counting Kit-8 (CCK-8) assay. The contents of IL-6, IL-8, TNF-α, IL-1β, and IL-10 in inoculated local tissue and the antigen-specific antibody titers in mouse serum samples were determined by ELISA. The target cells of IL-10 ASO were observed using immunofluorescent staining. The results showed that the specific antibody titer of ovalbumin (OVA), a model antigen, was increased 100-fold upon addition of IL-10 ASO as an adjuvant compared to that of OVA alone. IL-10 ASO showed an immunopotentiation efficacy similar to that of Freund’s incomplete adjuvant, with no detectable cell or tissue toxicity. In vitro and in vivo experiments confirmed that IL-10 ASO enhances immune responses by temporarily suppressing IL-10 expression from local dendritic cells and consequently promoting T cell proliferation. In conclusion, IL-10 ASO significantly enhances immune responses against co-delivered vaccine antigens with high efficacy and low toxicity. It has the potential to be developed into a safe and efficient immune adjuvant.     

Effect of immune regulatory pathways after immunization with GMZ2 malaria vaccine candidate in healthy lifelong malaria-exposed adults

BackgroundDespite appreciable immunogenicity in malaria-naive populations, many candidate malaria vaccines are considerably less immunogenic in malaria-exposed populations. This could reflect induction of immune regulatory mechanisms involving Human Leukocyte Antigen G (HLA-G), regulatory T (Treg), and regulatory B (Breg) cells. Here, we addressed the question whether there is correlation between these immune regulatory pathways and both plasmablast frequencies and vaccine-specific IgG concentrations.MethodsFifty Gabonese adults with lifelong exposure to Plasmodium spp were randomized to receive three doses of either 30 µg or 100 µg GMZ2-CAF01, or 100 µg GMZ2-alum, or control vaccine (rabies vaccine) at 4-week intervals. Only plasma and peripheral blood mononuclear cells isolated from blood samples collected before (D0) and 28 days after the third vaccination (D84) of 35 participants were used to measure sHLA-G levels and anti-GMZ2 IgG concentrations, and to quantify Treg, Breg and plasmablast cells. Vaccine efficacy was assessed using controlled human malaria infection (CHMI) by direct venous inoculation of Plasmodium falciparum sporozoites (PfSPZ Challenge).ResultsThe sHLA-G concentration increased from D0 to D84 in all GMZ2 vaccinated participants and in the control group, whereas Treg frequencies increased only in those receiving 30 µg or 100 µg GMZ2-CAF01. The sHLA-G level on D84 was associated with a decrease of the anti-GMZ2 IgG concentration, whereas Treg frequencies on D0 or on D84, and Breg frequency on D84 were associated with lower plasmablast frequencies. Importantly, having a D84:D0 ratio of sHLA-G above the median was associated with an increased risk of P. falciparum infection after sporozoites injection.ConclusionRegulatory immune responses are induced following immunization. Stronger sHLA-G and Treg immune responses may suppress vaccine induced immune responses, and the magnitude of the sHLA-G response increased the risk of Plasmodium falciparum infection after CHMI. These findings could have implications for the design and testing of malaria vaccine candidates in semi-immune individuals.     

Evaluation of BioThrax® and AV7909 anthrax vaccines in adults 66 years of age or older

BackgroundMultiple Anthrax vaccines are licensed or in development for post-exposure prophylaxis in individuals 18 to 65 years of age. No information exists on anthrax vaccines in populations over the age of 65. It is critical that we assess the capacity of anthrax vaccines to generate a protective immune response in older individuals. In this study, we compared BioThrax® to a formulation containing a CpG adjuvant (AV7909).MethodsWe conducted a Phase 2 clinical study to evaluate safety and immunogenicity of three vaccination schedules of the AV7909 vaccine candidate and one vaccination schedule of BioThrax® vaccine in adults over 65 years of age. A total of 305 subjects were enrolled to assess safety and immunogenicity by seroprotection rates, toxin neutralizing antibody titers, and anti-Protective Antigen ELISA titers.ResultsCompared to BioThrax, AV7909 elicited a more robust immune response in older subjects, especially with three doses of AV7909 at Days 1, 15, and 29, or two doses at Days 1 and 29. These trends were true with both seroprotection rates as defined by the percentage of subjects with 50 percent neutralization factors greater than 0.56, and geometric mean antibody titers. The responses to both AV7909 and BioThax were lower in older subjects compared to those aged 18–50.ConclusionThe immunogenicity data suggest that the CpG adjuvant in the AV7909 vaccine helps to elicit a more robust immune response in subjects over the age of 65. Alternative dosing strategies may be considered in this population given the high seroprotection rates with Day 1 and 29, or Day 1, 15, and 29 regimens.Trial Registration: clinicaltrials.gov Identifier: NCT03518125.