Antigen-specific Antibody Titers Research Articles

The aged microenvironment impairs BCL6 and CD40L induction in CD4+ T follicular helper cell differentiation.

Weakened germinal center responses by the aged immune system result in diminished immunity against pathogens and reduced efficacy of vaccines. Prolonged contacts between activated B cells and CD4+ T cells are crucial to germinal center formation and T follicular helper cell (Tfh) differentiation, but it is unclear how aging impacts the quality of this interaction. Peptide immunization confirmed that aged mice have decreased expansion of antigen-specific germinal center B cells and reduced antibody titers. Furthermore, aging was associated with accumulated Tfh cells, even in naïve mice. Despite increased numbers, aged Tfh had reduced expression of master transcription factor BCL6 and increased expression of the ectonucleotidase CD39. Invitro activation revealed that proliferative capacity was maintained in aged CD4+ T cells, but not the costimulatory molecule CD40L. When activated invitro by aged antigen-presenting cells, young CD4+ naïve T cells generated reduced numbers of activated cells with upregulated CD40L. To determine the contribution of cell-extrinsic influences on antigen-specific Tfh induction, young, antigen-specific B and CD4+ T cells were adoptively transferred into aged hosts prior to peptide immunization. Transferred cells had reduced expansion and differentiation into germinal center B cell and Tfh and reduced antigen-specific antibody titers when compared to young hosts. Young CD4+ T cells transferred aged hosts differentiated into Tfh cells with reduced PD-1 and BCL6 expression, and increased CD39 expression, though they maintained their mitochondrial capacity. These results highlight the role of the lymphoid microenvironment in modulating CD4+ T cell differentiation, which contributes to impaired establishment and maintenance of germinal centers.

Differences in Characteristics Between Patients Who Met or Partly Met the Diagnostic Criteria for Food Protein-Induced Enterocolitis Syndrome (FPIES)

Some patients with food protein-induced enterocolitis (FPIES)-like allergy do not completely fulfill the diagnostic criteria of the international consensus guideline for FPIES. However, it is unclear whether such FPIES-like patients represent a completely different population from FPIES. This study aimed to clarify differences in characteristics between patients with FPIES who fully met diagnostic criteria and those who partly met them. This was a cross-sectional study using data at the time of registration in multicenter, prospective studies of patients with FPIES in Japan. Children who had delayed emesis within 1 to 4 hours and/or diarrhea within 5 to 10 hours after ingestion of food were recruited between March 2020 and February 2022. We examined their compatibility with the diagnostic criteria of the international consensus guideline and their detailed clinical characteristics, including trigger foods, the serving size that elicited symptoms, and antigen-specific IgE antibody titers. Of the 225 patients with FPIES, 140 fully met the diagnostic criteria whereas 79 patients did not fully meet them but demonstrated reproducible symptoms. The frequencies of pallor, lethargy, and diarrhea were significantly higher in those who met the criteria fully, whereas the age at onset, trigger foods, comorbidity, and perinatal information were comparable. Analysis of patients with FPIES to hen's egg revealed significantly higher levels of egg white- and egg yolk-specific IgE in patients who partly met criteria, whereas the serving size eliciting symptoms was comparable. Patients who partly met the diagnostic criteria may have a milder phenotype of FPIES, but this needs to be validated in further studies using biomarkers reflecting the pathophysiology.

Immunophenotypic predictors of influenza vaccine immunogenicity in pediatric hematopoietic cell transplant recipients

AbstractPediatric hematopoietic cell transplant (HCT) recipients exhibit poor serologic responses to influenza vaccination early after transplant. To facilitate the optimization of influenza vaccination timing, we sought to identify B- and T-cell subpopulations associated with influenza vaccine immunogenicity in this population. We used mass cytometry to phenotype peripheral blood mononuclear cells collected from pediatric HCT recipients enrolled in a multicenter influenza vaccine trial comparing high- and standard-dose formulations over 3 influenza seasons (2016-2019). We fit linear regression models to estimate relationships between immune cell subpopulation numbers before vaccination and prevaccination to postvaccination geometric mean fold rises in antigen-specific (A/H3N2, A/H1N1, and B/Victoria) serum hemagglutination inhibition antibody titers (28-42 days, and ∼6 months after 2 doses). For cell subpopulations identified as predictive of a response to all 3 antigens, we conducted a sensitivity analysis including time after transplant as an additional covariate. Among 156 HCT recipients, we identified 33 distinct immune cell subpopulations; 7 significantly predicted responses to all 3 antigens 28 to 42 days after a 2-dose vaccine series, irrespective of vaccine dose. We also found evidence that baseline absolute numbers of naïve B cells, naïve CD4+ T cells, and circulating T follicular helper cells predicted peak and sustained vaccine-induced titers irrespective of dose or timing of posttransplant vaccine administration. In conclusion, several B- and T-cell subpopulations predicted influenza vaccine immunogenicity in pediatric HCT recipients. This study provides insights into the immune determinants of vaccine responses and may help guide the development of tailored vaccination strategies for this vulnerable population.

Lipid nanoparticle-encapsulated DNA vaccine robustly induce superior immune responses to the mRNA vaccine in Syrian hamsters

DNA vaccines for infectious diseases and cancer have been explored for years. To date, only one DNA vaccine (ZyCoV-D) has been authorized for emergency use in India. DNA vaccines are inexpensive and long-term thermostable, however, limited by the low efficiency of intracellular delivery. The recent success of mRNA/lipid nanoparticle (LNP) technology in the COVID-19 pandemic has opened a new application for nucleic acid-based vaccines. Here, we report that plasmid encoding a trimeric spike protein with LNP delivery (pTS/LNP), similar to those in Moderna's COVID-19 vaccine, induced more effective humoral responses than naked pTS or pTS delivered via electroporation. Compared to TSmRNA/LNP, pTS/LNP immunization induced a comparable level of neutralizing antibody titers and significant Th1-biased immunity in mice; it also prolonged the maintenance of higher antigen-specific IgG and neutralizing antibody titers in hamsters. Importantly, pTS/LNP immunization exhibits enhanced cross-neutralizing activity against SARS-CoV-2 variants and protects hamsters from the challenge of SARS-CoV-2 (Wuhan strain and omicron BA.1 variant). This study indicates that pDNA/LNPs as a promising platform could be a next-generation vaccine technology.

Immunogenicity of Escherichia coli Outer Membrane Vesicles: Elucidation of Humoral Responses against OMV-Associated Antigens.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have emerged as a novel and flexible vaccine platform. OMVs can be decorated with foreign antigens and carry potent immunostimulatory components. Therefore, after their purification from the culture supernatant, they are ready to be formulated for vaccine use. It has been extensively demonstrated that immunization with engineered OMVs can elicit excellent antibody responses against the heterologous antigens. However, the definition of the conditions necessary to reach the optimal antibody titers still needs to be investigated. Here, we defined the protein concentrations required to induce antigen-specific antibodies, and the amount of antigen and OMVs necessary and sufficient to elicit saturating levels of antigen-specific antibodies. Since not all antigens can be expressed in OMVs, we also investigated the effectiveness of vaccines in which OMVs and purified antigens are mixed together without using any procedure for their physical association. Our data show that in most of the cases OMV-antigen mixtures are very effective in eliciting antigen-specific antibodies. This is probably due to the capacity of OMVs to "absorb" antigens, establishing sufficiently stable interactions that allow antigen-OMV co-presentation to the same antigen presenting cell. In those cases when antigen-OMV interaction is not sufficiently stable, the addition of alum to the formulation guarantees the elicitation of high titers of antigen-specific antibodies.

Lipid Nanoparticle Delivery Alters the Adjuvanticity of the TLR9 Agonist CpG by Innate Immune Activation in Lymphoid Tissue.

Pharmacological strategies to activate innate immune cells are of great relevance in the context of vaccine design and anticancer immune therapy, to mount broad immune responses able to clear infection and malignant cells. Synthetic CpG oligodeoxynucleotides (CpG-ODNs) are short single-stranded DNA molecules containing unmethylated CpG dinucleotides and a phosphorothioate backbone. Class B CpG ODNs activate robust innate immune responses through a TLR9-dependent NF-κB signaling pathway. This feature is attractive to exploit in the context of vaccine design and cancer immunotherapy. Soluble CpG-ODNs cause hepatic toxicity, which reduces its therapeutic applicability. The formulation of class B CpG ODN1826 in lipid nanoparticles (LNPs) containing an ionizable cationic lipid that complexes CpG through electrostatic interaction is reported. Upon local administration, LNP-formulated CpG drains to lymph nodes and triggers robust innate immune activation. Unformulated, soluble, CpG, by contrast, is unable to induce robust innate activation in draining lymph nodes and is distributed systemically. In a vaccination setting, LNP-formulated CpG, admixed with a protein antigen, induces higher antigen-specific antibody titers and T cell responses than antigen admixed with unformulated soluble CpG.

Evaluation and comparison of immune responses induced by two Mpox mRNA vaccine candidates in mice.

The epidemic of Mpox virus (MPXV) from May 2022 was once declared as a Public Health Emergency of International Concern by the World Health Organization. Vaccines play an important role in prevention of infectious diseases, and mRNA vaccine technology was proved to be a safe and effective platform with successful application in defense of coronavirus disease 2019. In this study, based on A29L, M1R, A35R, and B6R of MPXV, we developed two MPXV mRNA vaccine candidates, designated as MPXfus and MPXmix. The MPXfus was one-component, in which these four antigen proteins were linked in tandem by flexible linker and encoded by an individual mRNA as a fusion protein. The MPXmix was multicomponent containing four mRNA, and each mRNA encoded one antigen protein respectively. Mice were immunized with equal quality of MPXfus or MPXmix, delivered by lipid nanoparticles for evaluation and comparison of the immune responses induced by these two MPXV vaccine candidates. Results of immune response analyses indicated that both MPXfus and MPXmix could elicit high-level of antigen-specific antibodies and robust cellular immune response in mice. Moreover, results of virus neutralization assays suggested that sera from MPXfus- or MPXmix-immunized mice possessed high neutralizing activities against vaccinia virus. In addition, titers of antigen-specific antibody, levels of cellular immune response, and activities of neutralizing antibody against vaccinia virus induced by MPXfus and MPXmix presented no significant difference. In summary, this study provides valuable insights for further clinical development of one-component and multicomponent mRNA vaccine candidates for the prevention of MPXV and other orthomyxoviruses.

Immune Response of Inactivated Rabies Vaccine Inoculated via Intraperitoneal, Intramuscular, Subcutaneous and Needle-Free Injection Technology-Based Intradermal Routes in Mice.

Inoculation routes may significantly affect vaccine performance due to the local microenvironment, antigen localization and presentation, and, therefore, final immune responses. In this study, we conducted a head-to-head comparison of immune response and safety of inactivated rabies vaccine inoculated via intraperitoneal (IP), intramuscular (IM), subcutaneous (SC) and needle-free injection technology-based intradermal (ID) routes in ICR mice. Immune response was assessed in terms of antigen-specific antibodies, antibody subtypes and neutralizing antibodies for up to 28 weeks. A live rabies virus challenge was also carried out to evaluate vaccine potency. The dynamics of inflammatory cell infiltration at the skin and muscle levels were determined via histopathological examination. The kinetics and distribution of a model antigen were also determined by using in vivo fluorescence imaging. Evidence is presented that the vaccine inoculated via the ID route resulted in the highest antigen-specific antibody and neutralizing antibody titers among all administration routes, while IP and IM routes were comparable, followed by the SC route. Antibody subtype analysis shows that the IP route elicited a Th1-biased immune response, while SC and IM administration elicited a prominent Th2-type immune response. Unexpectedly, the ID route leads to a balanced Th1 and Th2 immune response. In addition, the ID route conferred effective protection against lethal challenge with 40 LD50 of the rabies CVS strain, which was followed by IP and IM routes. Moreover, a one-third dose of the vaccine inoculated via the ID route provided comparable or higher efficacy to a full dose of the vaccine via the other three routes. The superior performance of ID inoculation over other routes is related to longer local retention at injection sites and higher lymphatic drainage. Histopathology examination reveals a transient inflammatory cell infiltration at ID and IM injection sites which peaked at 48 h and 24 h, respectively, after immunization, with all side effects disappearing within one week. These results suggest that needle-free injection technology-based ID inoculation is a promising strategy for rabies vaccination in regard to safety and efficacy.

Co-delivery of a trimeric spike DNA and protein vaccine with aluminum hydroxide enhanced Th1-dominant humoral and cellular immunity against SARS-CoV-2.

Protein subunit vaccines have been used as prophylactic vaccines for a long time. The well-established properties of these vaccines make them the first choice for the coronavirus disease 2019 (COVID-19) outbreak. However, it is not easy to develop a protein vaccine that induces cytotoxic T lymphocyte responses and requires a longer time for manufacturing, which limits the usage of this vaccine type. Here, we report the combination of a recombinant spike (S)-trimer protein with a DNA vaccine-encoded S protein as a novel COVID-19 vaccine. The recombinant S protein was formulated with different adjuvants and mixed with the DNA plasmid before injection. We found that the recombinant S protein formulated with the adjuvant aluminum hydroxide and mixed with the DNA plasmid could enhance antigen-specific antibody titers, neutralizing antibody titers. We further evaluated the IgG2a/IgG1 isotype and cytokine profiles of the specific boosted T-cell response, which indicated that the combined vaccine induced a T-helper 1 cell-biased immune response. Immunized hamsters were challenged with severe acute respiratory syndrome coronavirus 2, and the body weight of the hamsters that received the recombinant S protein with aluminum hydroxide and/or the DNA plasmid was not reduced. Alternatively, those that received control or only the DNA plasmid immunization were reduced. Interestingly, after the third day of the viral load in the lungs, the viral challenge could not be detected in hamsters immunized with the recombinant S protein in aluminum hydroxide mixed with DNA(tissue culture infectious dose < 10). The viral load in the lungs was 109 , 106 , and 107 for the phosphate-buffered saline, protein in aluminum hydroxide, and DNA-only immunizations, respectively. These results indicated that antiviral mechanisms neutralizing antibodies play important roles. Furthermore, we found that the combination of protein and DNA vaccination could induce relatively strong CD8+ T-cell responses. In summary, the protein subunit vaccine combined with a DNA vaccine could induce strong CD8+ T-cell responses to increase antiviral immunity for disease control.

Lipidated brartemicin adjuvant p-C18Brar is a promising α,α′-trehalose 6,6′-dilipid for use in ovine pneumonia vaccines

Abstract Ovine pneumonia is a disease in sheep that is associated with major animal welfare issues and economic losses and for which there is no effective vaccine. We tested the adjuvanticity of our most promising α,α′-trehalose 6,6′-glycolipids, lipidated brartemicin adjuvants p-C18Brar (3), o-C18Brar (4), and amide-TDB (5) in vaccines for ovine pneumonia containing Mannheimia haemolytica and Mycoplasma ovipneumoniae whole cell antigens. p-C18Brar (3) and o-C18Brar (4) led to strong antigen-specific IgG antibody titres that were better than those elicited by the prototypical α,α′-trehalose glycolipid trehalose dibehenate (TDB, 2) and amide-TDB (5). T-cell responses, as determined by measuring IFN-γ and IL-17A production from antigen-stimulated whole blood cultures, revealed that p-C18Brar (3), but not TDB (2), o-C18Brar (4), or amide-TDB (5), led to statistically significant increases in these cytokines. We then optimised the synthesis of p-C18Brar (3) (3 steps, 72 % overall yield) and undertook further vaccination studies to determine the optimal dose of p-C18Brar (3) that would be used for future large scale ovine pneumonia field trials. At a dose of 3.75 mg per vaccine, the adjuvanticity of p-C18Brar (3), as measured by levels of anti-M. haemolytica IgG antibody and T-cell responses (IFN-γ and IL-17A) was better than that elicited by the commercially available adjuvant Quil-A, and had reduced reactogenicity. Taken together, the excellent immunological profile of p-C18Brar (3) and its ease and efficiency of synthesis makes it an attractive adjuvant for use in veterinary vaccines.

Sulfated Lactosyl Archaeol Archaeosome-Adjuvanted Vaccine Formulations Targeting Rabbit Hemorrhagic Disease Virus Are Immunogenic and Efficacious.

Vaccines play an important role in maintaining human and animal health worldwide. There is continued demand for effective and safe adjuvants capable of enhancing antigen-specific responses to a target pathogen. Rabbit hemorrhagic disease virus (RHDV) is a highly contagious calicivirus that often induces high mortality rates in rabbits. Herein, we evaluated the activity of an experimental sulfated lactosyl archaeol (SLA) archaeosome adjuvant when incorporated in subunit vaccine formulations targeting RHDV. The subunit antigens consisted of RHDV-CRM197 peptide conjugates or recombinant RHDV2 VP60. SLA was able to enhance antigen-specific antibody titers and cellular responses in mice and rabbits. Three weeks following immunization, antigen-specific antibody levels in rabbits vaccinated with RHDV2 VP60 + SLA were significantly higher than those immunized with antigen alone, with geomean titers of 7393 vs. 117. In addition, the SLA-adjuvanted VP60-based formulations were highly efficacious in a rabbit RHDV2 challenge model with up to 87.5% animals surviving the viral challenge. These findings demonstrate the potential utility of SLA adjuvants in veterinary applications and highlight its activity in different types of mammalian species.

Alterations in germinal center formation and B cell activation during severe Orientia tsutsugamushi infection in mice.

Scrub typhus is a poorly studied but life-threatening disease caused by the intracellular bacterium Orientia tsutsugamushi (Ot). Cellular and humoral immunity in Ot-infected patients is not long-lasting, waning as early as one-year post-infection; however, its underlying mechanisms remain unclear. To date, no studies have examined germinal center (GC) or B cell responses in Ot-infected humans or experimental animals. This study was aimed at evaluating humoral immune responses at acute stages of severe Ot infection and possible mechanisms underlying B cell dysfunction. Following inoculation with Ot Karp, a clinically dominant strain known to cause lethal infection in C57BL/6 mice, we measured antigen-specific antibody titers, revealing IgG2c as the dominant isotype induced by infection. Splenic GC responses were evaluated by immunohistology, co-staining for B cells (B220), T cells (CD3), and GCs (GL-7). Organized GCs were evident at day 4 post-infection (D4), but they were nearly absent at D8, accompanied by scattered T cells throughout splenic tissues. Flow cytometry revealed comparable numbers of GC B cells and T follicular helper (Tfh) cells at D4 and D8, indicating that GC collapse was not due to excessive death of these cell subtypes at D8. B cell RNAseq analysis revealed significant differences in expression of genes associated with B cell adhesion and co-stimulation at D8 versus D4. The significant downregulation of S1PR2 (a GC-specific adhesion gene) was most evident at D8, correlating with disrupted GC formation. Signaling pathway analysis uncovered downregulation of 71% of B cell activation genes at D8, suggesting attenuation of B cell activation during severe infection. This is the first study showing the disruption of B/T cell microenvironment and dysregulation of B cell responses during Ot infection, which may help understand the transient immunity associated with scrub typhus.

Unmasking Arterial-resident Autoreactive B cells involved in Atherosclerosis and Peripheral Artery Disease Progression

Abstract Atherosclerosis activates the immune system, including B cells which secrete antibodies that bind antigens released by necrotic cells within plaques and may promote inflammation. To identify these antibodies, we isolated activated B cells from atherosclerotic ApoE-deficient mice aortas and human tibial arteries with peripheral artery disease post-amputation. Single-cell sequencing of the B cell receptors identified 35 mouse and 48 human overrepresented disease-relevant heavy and light chain pairings, which were sequentially expressed as antibody. Epitope determination was performed by human proteome array, and 14 mouse and 21 human protein targets were found with significant affinity and specificity. Mouse antigens were scrutinized by immunohistochemistry to confirm presence in inflamed plaques, and expression of these proteins were used to interrogate autoreactive antibody titers in diseased sera by ELISA. ApoE-deficient mice were immunized with the 6 most promising antigens. Serum antibody measurement showed strong, rapid, and early onset of antigen-specific antibody responses in the immunized mice compared to an adjuvant only control group. Strikingly, we saw decreased plaque accumulation in the immunized animals, suggesting antigen responses may clear pathogenic antigens before disease develops. Likewise, human antigen ELISAs comparing antigen-specific serum antibody titers to patient Framingham score for cardiovascular risk supported a role for two antigens in disease. To confirm these antigens as potential biomarkers, we will characterize the emergence of their antibody counterparts using a historical library of serum samples from individuals enrolled in the Baltimore Longitudinal Study on Aging. This work is directly funded by the National Institutes of Health – National Institute on Aging’s Intramural Research Program in the Lab of Molecular Biology and Immunology

MRNA-adjuvanted Influenza A Vaccine Can Induce High Cellular Immunity and Cross Reactivity in Mice

Abstract In recent years, the great success of mRNA-based vaccines for SARS-CoV-2 revealed a potential future for safe and effective mRNA vaccine designs. Given the flexibility and accessibility of mRNA designs, adjuvants in mRNA forms may be applied to enhance immune response and generate better cross-protection. In this study, we fabricated an Influenza mRNA lipid nanoparticle (LNP) vaccine with Influenza A/Aichi/2/1968 (Aichi, H3N2) Hemagglutinin (HA) mRNA and cytokine-adjuvant mRNA (GIFT4 or CCL27). We found that two doses of immunization with mRNA-adjuvanted LNPs induced increased antigen-specific antibody titer and effective protection against Influenza A viruses in mice. The supplement of cytokine mRNAs (GIFT4 or CCL27) provided more robust immunogenicity, including more IL-4 and IFN-γ secreting T cells in the spleen and more IgG and IgA-secreting B cells in the bone marrow. The cytokine mRNA adjuvants also induced early activation of T and B cells in germinal centers. Thus, GIFT4 and CCL27 mRNAs are potential adjuvants for Influenza A mRNA vaccine to elicit higher and broader immune responses in mice.

Method of manufacturing CAF®09 liposomes affects immune responses induced by adjuvanted subunit proteins

The ability to induce antigen-specific CD4+ and CD8+T-cell responses is one of the fundamental requirements when developing new efficacious vaccines against challenging infectious diseases and cancer. However, no adjuvants are currently approved for human subunit vaccines that induce T-cell immunity. Here, we incorporated a Toll-like receptor 4 agonist, i.e., the ionizable lipidoid L5N12, in the liposomal cationic adjuvant formulation 09 (CAF®09), and found that modified CAF®09 liposomes possess preserved adjuvant function as compared to unmodified CAF®09. CAF®09 consists of the cationic lipid dimethyldioctadecylammonium (DDA), monomycoloyl glycerol analogue 1 (MMG-1), and polyinosinic:polycytidylic acid [poly(I:C)]. By using the microfluidic mixing technology for liposome preparation, we gradually replaced DDA with L5N12, while keeping the molar ratios of MMG-1 and poly(I:C) constant. We found that this type of modification resulted in colloidally stable liposomes, which were significantly smaller and displayed reduced surface charge as compared to unmodified CAF®09, prepared by using the conventional thin film method. We showed that incorporation of L5N12 decreases the membrane rigidity of CAF®09 liposomes. Furthermore, vaccination with antigen adjuvanted with L5N12-modified CAF®09 or antigen adjuvanted with unmodified CAF®09, respectively, induced comparable antigen-specific serum antibody titers. We found that antigen adjuvanted with L5N12-modified CAF®09 induced antigen-specific effector and memory CD4+ and CD8+T-cell responses in the spleen comparable to those induced when unmodified CAF®09 was used as adjuvant. However, incorporating L5N12 did not have a synergistic immunopotentiating effect on the antibody and T-cell responses induced by CAF®09. Moreover, vaccination with antigen adjuvanted with unmodified CAF®09, which was manufactured by using microfluidic mixing, induced significantly lower antigen-specific CD4+ and CD8+T-cell responses than vaccination with antigen adjuvanted with unmodified CAF®09, which was prepared by using the thin film method. These results show that the method of manufacturing affects CAF®09 liposome adjuvanted antigen-specific immune responses, which should be taken into consideration when evaluating immunogenicity of subunit protein vaccines.

Recombinant full-length Bacillus Anthracis protective antigen and its 63 kDa form elicits protective response in formulation with addavax.

Bacillus anthracis is the causative agent for the lethal disease anthrax, primarily affecting animals and humans in close contact with an infected host. The pathogenicity of B. anthracis is attributed to the secreted exotoxins and their outer capsule. The host cell-binding exotoxin component "protective antigen" (PA) is reported to be a potent vaccine candidate. The aim of our study is to produce several PA constructs and analyze their vaccine potential. We have designed the various subunit, PA-based recombinant proteins, i.e., full-length Protective antigen (PA-FL), C-terminal 63 kDa fragment (PA63), Protective antigen domain 1-domain 4 chimeras (PA-D1-4) and protective antigen domain 4 (PA-D4) and analyzed their vaccine potential with different human-compatible adjuvants in the mouse model. We have optimized the process and successfully expressed our recombinant antigens as soluble proteins, except full-length PA. All the recombinant antigen formulations with three different adjuvants i.e., Addavax, Alhydrogel, and Montanide ISA 720, were immunized in different mouse groups. The vaccine efficacy of the formulations was analyzed by mouse serum antigen-specific antibody titer, toxin neutralization assay, and survival analysis of mouse groups challenged with a lethal dose of B. anthracis virulent spores. We have demonstrated that the PA-FL addavax and PA63 addavax formulations were most effective in protecting spore-challenged mice and serum from the mice immunized with PAFL addavax, PA-FL alhydrogel, PA63 addavax, and PA63 alhydrogel formulations were equivalently efficient in neutralizing the anthrax lethal toxin. The higher levels of serum Th1, Th2, and Th17 cytokines in PA-FL addavax immunized mice correspond to the enhanced protection provided by the formulation in challenged mice. We have demonstrated that the PA-FL addavax and PA63 addavax formulations exhibit equivalent efficiency as vaccine formulation both in a mouse model of anthrax and mammalian cell lines. However, PA63 is a smaller antigen than PA-FL and more importantly, PA63 is expressed as a soluble protein in E. coli, which imparts a translational advantage to PA63-based formulation. Thus, the outcome of our study has significant implications for the development of protective antigen-based vaccine formulations for human use against the lethal disease anthrax.

PS-B04-12: DEVELOPMENT OF VACCINE AGAINST CONNECTIVE TISSUE GROWTH FACTOR TO PREVENT THE PROGRESSION OF RENAL FIBROSIS

Objective: Hypertension is closely related with chronic kidney disease (CKD); Hypertension and CKD can cause and exacerbate each other, forming a vicious cycle. There is a critical need for efficient treatment of CKD. Renal fibrosis is a final common pathway to end-stage renal disease, and connective tissue growth factor (CTGF) is a well-recognized central profibrotic factor in fibrosis of various organ systems. Here, we developed a novel peptide vaccine against CTGF to prevent the progression of renal fibrosis. Design and method: Among sequences of CTGF, the sequence common to mice and humans in the interaction region with transforming growth factor-β; (TGF-β;) and bone morphogenic proteins was selected as the epitope for the vaccine. The efficacy of this vaccine was verified in two renal fibrosis models using male C57BL/6J mice: adenine-induced CKD and unilateral ureteral obstruction (UUO) models. Moreover, the functions of CTGF vaccine-elicited IgG antibodies were evaluated in an in vitro study. Results: Three immunizations with this CTGF vaccine at 2-week intervals elicited serum IgG antibodies specifically binding to human full-length CTGF, and the antigen-specific antibody titers were maintained at high level for &gt; 30 weeks. In adenine-induced CKD model, histological analyses showed that immunization with the CTGF vaccine significantly attenuated renal interstitial fibrosis compared to the vehicle treatment (8.5 ± 0.8% vs 12.3 ± 0.8%; P &lt; 0.01). The CTGF vaccination also suppressed the increased mRNA expressions of collagen 1α and α-smooth muscle actin (α-SMA) in kidney tissue samples. Moreover, vaccinated mice showed lower levels of serum urea nitrogen (UN) and creatinine and lower urine albumin-creatinine ratio compared with vehicle-treated mice (UN: 77.7 ± 5.0 vs 104.0 ± 6.8 mg/dL; P &lt; 0.01. creatinine: 0.42 ± 0.02 vs 0.51 ± 0.04 mg/dL; P = 0.06. albumin-creatinine ratio: 74.7 ± 13.8 vs 132.0 ± 15.3 μg/mg; P &lt; 0.05). In UUO model, the CTGF vaccination also ameliorated the expansion of the interstitial fibrotic areas (7.7 ± 0.4 vs 9.8 ± 0.7%; P &lt; 0.05). In an in vitro study, CTGF vaccine-elicited IgG antibodies efficiently suppressed CTGF-induced- and TGF-β;-induced α-SMA expression in cultured kidney fibroblasts. Conclusions: The CTGF vaccine has the potential to prevent the progression of renal fibrosis and delay the deterioration of renal function with a low frequency of administration. Thus, this CTGF vaccine could be a promising preventive strategy for CKD patients.

112. Interim Results From a Phase 2, Randomized, Observer-Blind, Placebo-Controlled, Dose-Finding Trial of an mRNA-Based Cytomegalovirus Vaccine in Healthy Adults

Abstract Background Cytomegalovirus (CMV) is the most common congenital viral infection and can cause severe long-term health consequences, including hearing loss and neurodevelopmental delay. A safe and effective method for prevention of CMV infection is an unmet need and public health priority. An mRNA-based vaccine against CMV, mRNA-1647, is in development and consists of 6 mRNA sequences encoding 2 CMV antigens (glycoprotein B and the pentameric glycoprotein complex) in a lipid nanoparticle formulation. Methods In this Phase 2, randomized, placebo-controlled, observer-blind, dose-finding trial, safety and immunogenicity of mRNA-1647 was evaluated in healthy adults aged 18 to 40 years (NCT04232280). In Part 1, CMV-seronegative and CMV-seropositive men and women were randomized 3:1 to receive mRNA-1647 (doses of 50, 100, or 150 µg) or placebo at Months 0, 2, and 6. The 100-µg dose was chosen for Part 2. In Part 2, CMV-seronegative and CMV-seropositive women were randomized 3:1 to receive mRNA-1647 100 µg or placebo at Months 0, 2, and 6. Safety endpoints were solicited local and systemic adverse reactions, unsolicited adverse events (AEs), and medically attended AEs through 7 days, 28 days, and 6 months after vaccination, respectively, and serious AEs throughout the study. Humoral immunogenicity endpoints were antigen-specific binding antibody titers and neutralizing antibody titers against epithelial cell infection and against fibroblast infection. Results In Parts 1 and 2, 252 and 63 participants were randomized, respectively. Interim analysis (IA) of Part 1 through 1 month after Dose 3 indicated that mRNA-1647 100 µg was generally well-tolerated, induced robust antibody responses in CMV-seronegative participants, and boosted antibody titers in CMV-seropositive participants. An additional IA of Part 1 through end of study and Part 2 through 1 month after Dose 3 showed no notable differences in the safety profile compared with the previous IA (Part 1, 1 month after Dose 3); immunogenicity data for this additional IA is being generated. Conclusion Available data from this Phase 2 trial suggest that mRNA-1647 100 µg was immunogenic in CMV-seronegative and CMV-seropositive participants and was generally well-tolerated. The mRNA-1647 candidate vaccine is being evaluated in a Phase 3 trial. Disclosures Lori Panther, MD, MPH, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Richard Leggett, DO, Crossroads Clinical Research: Contract employee James Peterson, MD, Moderna, Inc.: Received payment as a study investigator Jiang Lin, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Kai Wu, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Heather Lee, BS, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Roxane Hasselbeck, BA, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Andrew Natenshon, MA, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Jacqueline Miller, MD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds.

Pam2CSK4-adjuvanted SARS-CoV-2 RBD nanoparticle vaccine induces robust humoral and cellular immune responses.

As the global COVID-19 pandemic continues and new SARS-CoV-2 variants of concern emerge, vaccines remain an important tool for preventing the pandemic. The inactivated or subunit vaccines themselves generally exhibit low immunogenicity, which needs adjuvants to improve the immune response. We previously developed a receptor binding domain (RBD)-targeted and self-assembled nanoparticle to elicit a potent immune response in both mice and rhesus macaques. Herein, we further improved the RBD production in the eukaryote system by in situ Crispr/Cas9-engineered CHO cells. By comparing the immune effects of various Toll-like receptor-targeted adjuvants to enhance nanoparticle vaccine immunization, we found that Pam2CSK4, a TLR2/6 agonist, could mostly increase the titers of antigen-specific neutralizing antibodies and durability in humoral immunity. Remarkably, together with Pam2CSK4, the RBD-based nanoparticle vaccine induced a significant Th1-biased immune response and enhanced the differentiation of both memory T cells and follicular helper T cells. We further found that Pam2CSK4 upregulated migration genes and many genes involved in the activation and proliferation of leukocytes. Our data indicate that Pam2CSK4 targeting TLR2, which has been shown to be effective in tuberculosis vaccines, is the optimal adjuvant for the SARS-CoV-2 nanoparticle vaccine, paving the way for an immediate clinical trial.

Formulation, inflammation, and RNA sensing impact the immunogenicity of self-amplifying RNA vaccines

To be effective, RNA vaccines require both in situ translation and the induction of an immune response to recruit cells to the site of immunization. These factors can pull in opposite directions with the inflammation reducing expression of the vaccine antigen. We investigated how formulation affects the acute systemic cytokine response to a self-amplifying RNA (saRNA) vaccine. We compared a cationic polymer (pABOL), a lipid emulsion (nanostructured lipid carrier, NLC), and three lipid nanoparticles (LNP). After immunization, we measured serum cytokines and compared the response to induced antibodies against influenza virus. Formulations that induced a greater cytokine response induced a greater antibody response, with a significant correlation between IP-10, MCP-1, KC, and antigen-specific antibody titers. We then investigated how innate immune sensing and signaling impacted the adaptive immune response to vaccination with LNP-formulated saRNA. Mice that lacked MAVS and are unable to signal through RIG-I-like receptors had an altered cytokine response to saRNA vaccination and had significantly greater antibody responses than wild-type mice. This indicates that the inflammation induced by formulated saRNA vaccines is not solely deleterious in the induction of antibody responses and that targeting specific aspects of RNA vaccine sensing might improve the quality of the response.