Montanide ISA Research Articles

CTIM-01. OUTCOMES AND IMMUNE RESPONSES AFTER PEPTIDE VACCINATION TARGETING HUMAN CYTOMEGALOVIRUS ANTIGEN PP65 IN CHILDREN AND YOUNG ADULTS WITH RECURRENT HIGH-GRADE GLIOMA AND MEDULLOBLASTOMA: RESULTS OF A PHASE 1 TRIAL

Abstract INTRODUCTION The human cytomegalovirus (CMV) antigen pp65 is ubiquitously expressed in high-grade glioma (HGG) and medulloblastoma but not in adjacent brain. The primary objective of this phase I trial (NCT03299309) was to assess the safety and feasibility of a novel peptide vaccine targeting pp65 (PEP-CMV) in children/young adults with recurrent medulloblastoma and HGG. METHODS PEP-CMV is comprised of a synthetic long peptide of 26 amino acids and is administered as an emulsion in Montanide ISA 51. Patients receive a 5-day course of temozolomide to induce lymphopenia, tetanus/diphtheria toxoid site preconditioning to promote dendritic cell migration, then PEP-CMV intradermally in the groin every two weeks for 3 doses, then monthly. RESULTS 42 patients were enrolled. The mean age was 23.2 ± 9.3 years. 55% of patients were male. The median KPS was 80. The median number of prior chemotherapy regimens was 4 (range 1-15). Of the 38 patients who received PEP-CMV, the maximum grade adverse events (AE) possibly, probably, or definitely related to PEP-CMV were: 17 (45%) Grade 1 AEs, 16 (42%) Grade 2 AEs, 2 (5%) Grade 3 AEs (encephalopathy and pyramidal tract syndrome), and one (3%) Grade 4 AE (cerebral edema). One patient developed cytokine release syndrome (Grade 2). The median PFS was 2.5 months (95% CI:2.2, 3.2) and median OS was 6.4 months (95% CI:3.4, 7.9). The 12-month OS was 26.6% (95% CI:14, 41.1%). Of the 21 patients with evaluable immune monitoring data (received ≥3 vaccines/known CMV status), T cell reactivity on IFNγ pp65 ELISpot was increased after PEP-CMV delivery (median 2 vs 22, P=0.01). PFS and OS were significantly better in patients with low levels of CD56brightCD16dim phenotype NK cells: P=0.029 and P=0.047, respectively. Patients with naïve CD8+ counts over the median had significantly better PFS (P=0.006). CONCLUSIONS PEP-CMV is well-tolerated and elicits an antigen-specific immune response in heavily pretreated, multiply recurrent patients with a 12-month OS of 26.6%.

Design of the conserved epitope peptide of SARS-CoV-2 spike protein as the broad-spectrum COVID-19 vaccine

Our previous study has found that monoclonal antibodies targeting a conserved epitope peptide spanning from residues 1144 to 1156 of SARS-CoV-2 spike (S) protein, namely S(1144–1156), can broadly neutralize all of the prevalent SARS-CoV-2 strains, including the wild type, Alpha, Epsilon, Delta, and Gamma variants. In the study, S(1144–1156) was conjugated with bovine serum albumin (BSA) and formulated with Montanide ISA 51 adjuvant for inoculation in BALB/c mice to study its potential as a vaccine candidate. Results showed that the titers of S protein-specific IgGs and the neutralizing antibodies in mouse sera against various SARS-CoV-2 variants, including the Omicron sublineages, were largely induced along with three doses of immunization. The significant release of IFN-γ and IL-2 was also observed by ELISpot assays through stimulating vaccinated mouse splenocytes with the S(1144–1156) peptide. Furthermore, the vaccination of the S(1143–1157)- and S(1142–1158)-EGFP fusion proteins can elicit more SARS-CoV-2 neutralizing antibodies in mouse sera than the S(1144–1156)-EGFP fusion protein. Interestingly, the antisera collected from mice inoculated with the S(1144–1156) peptide vaccine exhibited better efficacy for neutralizing Omicron BA.2.86 and JN.1 subvariants than Omicron BA.1, BA.2, and XBB subvariants. Since the amino acid sequences of the S(1144–1156) are highly conserved among various SARS-CoV-2 variants, the immunogen containing the S(1144–1156) core epitope can be designed as a broadly effective COVID-19 vaccine.Key points• Inoculation of mice with the S(1144–1156) peptide vaccine can induce bnAbs against various SARS-CoV-2 variants.• The S(1144–1156) peptide stimulated significant release of IFN-γ and IL-2 in vaccinated mouse splenocytes.• The S(1143–1157) and S(1142–1158) peptide vaccines can elicit more SARS-CoV-2 nAbs in mice.

AML-VAC-XS15-01: protocol of a first-in-human clinical trial to evaluate the safety, tolerability and preliminary efficacy of a multi-peptide vaccine based on leukemia stem cell antigens in acute myeloid leukemia patients.

Acute myeloid leukemia (AML) has a dismal prognosis, mostly due to minimal residual disease-driven relapse, making an elimination of persisting therapy-resistant leukemia progenitor/stem cells (LPCs) the main goal for novel therapies. Peptide-based immunotherapy offers a low-side-effect approach aiming to induce T cell responses directed against human leukocyte antigen (HLA) presented tumor antigens on malignant cells by therapeutic vaccination. Mass spectrometry-based analysis of the naturally presented immunopeptidome of primary enriched LPC and AML samples enabled the selection of antigens exclusively expressed on LPC/AML cells, which showed de novo induction and spontaneous memory T cell responses in AML patients, and whose presentation and memory T cell recognition was associated with improved disease outcome. Based on these data the therapeutic vaccine AML-VAC-XS15 was designed, comprising two mutated HLA class I-restricted peptides from the common AML-specific mutation in NPM1 and seven HLA class II-restricted peptides (six non-mutated high-frequent AML/LPC-associated antigens and one mutated peptide from the AML-specific mutation R140Q in IDH2), adjuvanted with the toll like receptor 1/2 ligand XS15 and emulsified in Montanide ISA 51 VG. A phase I open label clinical trial investigating AML-VAC-XS15 was designed, recruiting AML patients in complete cytological remission (CR) or CR with incomplete blood count recovery. Patients are vaccinated twice with a six-week interval, with an optional booster vaccination four months after 2nd vaccination, and are then followed up for two years. The trial's primary objectives are the assessment of the vaccine's immunogenicity, safety and toxicity, secondary objectives include characterization of vaccine-induced T cell responses and assessment of preliminary clinical efficacy. The AML-VAC-XS15-01 study was approved by the Ethics Committee of the Bavarian State medical association and the Paul-Ehrlich Institut (P01392). Clinical trial results will be published in peer-reviewed journals.

CT584 Is Not a Protective Vaccine Antigen against Respiratory Chlamydial Challenge in Mice.

Background:Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen in humans worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and decentralized production of recombinant protein vaccine antigens. Methods: Here, we use CFPS to produce the full-length putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for evaluation as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) (RIBM, Tsukuba, Japan) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four weeks later by an i.m. boost before respiratory challenge with 104 inclusion forming units (IFU) of Chlamydia muridarum. Results: Immunization with CT584 generated robust antibody responses but weak cell-mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lung weights, and the presence of high numbers of IFUs in the lungs. Conclusion: While CT584 was not a protective vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens make it an attractive technique for antigen production.

Evaluation of comparative effect between aluminum hydroxide gel and montanide (ISA 70) in potency and protection of locally prepared rabbit hemorrhagic disease virus 2 (RHDV2) vaccines in rabbits

BackgroundRabbit hemorrhagic disease (RHD) is an acute infectious disease that damages the rabbit industry by producing significant mortality rates in young and adult rabbits. RHD is better controlled by vaccination.ObjectiveThe current study's goal was to prepare and evaluate the immuno-enhancing effect of montanide ISA70 and aluminum hydroxide (Al(OH)3) gel incorporated within the inactivated RHDV2 vaccine and assess the vaccine's protective efficacy against the homologous and heterologous local RHDV2 strains in rabbits.MethodsInactivated RHDV vaccines were prepared using Montanide ISA70 oil or Al(OH)3 gel adjuvants and submitted to sterility, safety, and potency tests. 200 rabbits were equally divided into 4 groups: G1 (control), G2 (vaccinated with gel-incorporated vaccine), G3 (vaccinated with montanide-incorporated vaccine), and G4 (vaccinated with gel- and montanide-incorporated vaccines). Individual blood samples were collected from one week to six months from all groups. The vaccine's potency was measured by the HI test and protection percentage post challenge.ResultsData revealed slightly increasing HI titer means reaching the 1st peak at 4 weeks post-vaccination (7.33, 7.67, and 7.33 log2 in the 2nd, 3rd, and 4th groups, respectively), then slightly decreasing and peaked again, giving 9.33 log2 for the2nd group at 3 months post-vaccination (MPV), 10.67 log2 for 3rd the group, and 10.33 log2 for the 4th group at 5 months post-vaccination. Titer gradually decreased but remained protective. The protection rate ranged from 80–100% and 80–90% for homologous and heterologous local RHDV2 vaccines, respectively, within 3 weeks and 6 months post-challenge. The montanide oil RHDV2 vaccine induced better protection than the aluminum gel RHDV2 vaccine.ConclusionThe results demonstrated evidence of cross-protection between RHDV2 strains. The oil emulsion vaccine induced higher and longer-lasting antibody titers than those obtained with the RHDV2 aluminum gel vaccine.

A candidate tick-borne encephalitis virus vaccine based on virus-like particles induces specific cellular and humoral immunity in mice1

Tick-borne encephalitis (TBE) is an important zoonotic viral disease transmitted by ticks. In recent decades, global climate change has increased human exposure to ticks, and mortality rates have gradually risen. Effective vaccines are essential for controlling TBE as specific antiviral treatment is unavailable. Vaccine candidates based on virus-like particles (VLPs) have previously been demonstrated to be efficient in eliciting excellent immune responses against influenza virus and SARS-CoV-2. Here, we constructed TBE virus (TBEV) VLPs containing the envelope and membrane proteins derived from the Far Eastern TBEV strain (WH2012) using an insect cell-baculovirus expression system. Induction of immune responses was investigated in mice following intramuscular injection with the TBEV VLPs vaccine candidates formulated of Poly(I:C) & Montanide ISA 201VG combination adjuvants. Mice produced memory T-cells and serum-specific IgG antibodies that averaged up to 1:104.6 and remained at 1:104 (mean) for 24 weeks after three immunizations. TBEV VLPs vaccine was able to provide long-term antibody protection against TBEV, making it a promising subunit vaccine candidate for this disease.

Vaccination with a Lawsonia intracellularis subunit water in oil emulsion vaccine mitigated some disease parameters but failed to affect shedding

Lawsonia intracellularis is the causative agent of ileitis in swine that manifests as slower weight gain, mild or hemorrhagic diarrhea and/or death in severe cases. As an economically important swine pathogen, development of effective vaccines is important to the swine industry. In developing a subunit vaccine with three recombinant antigens - FliC, GroEL and YopN – we wanted to identify a formulation that would produce robust immune responses that reduce disease parameters associated with Lawsonia intracellularis infection. We formulated these three antigens with four adjuvants: Montanide ISA 660 VG, Montanide Gel 02 PR, Montanide IMS 1313 VG NST, and Montanide ISA 61 VG in an immunogenicity study. Groups vaccinated with formulations including Montanide ISA 660 VG or Montanide ISA 61 VG had significantly more robust immune responses than groups vaccinated with formulations including Montanide Gel 02 PR or Montanide IMS 1313 VG NST. In the challenge study, animals vaccinated with these antigens and Montanide ISA 61 VG had reduced lesion scores, reduced lesion lengths, and increased average daily gain, but no reduction in shedding relative to the control animals. This work shows that this vaccine formulation should be considered for future study in a field and performance trial.

IVAC-XS15-CLL01: personalized multi-peptide vaccination in combination with the TLR1/2 ligand XS15 in CLL patients undergoing BTK-inhibitor-based regimens.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia among adults in Western countries. Despite the introduction of targeted therapies, including first-line Bruton's tyrosine kinase inhibitor (BTKi) treatment, CLL remains largely incurable. Frequent disease relapses occur due to remaining treatment-resistant CLL cells, calling for novel therapies to eliminate minimal residual disease (MRD). Peptide-based vaccination targeting human leucocyte antigen (HLA)-presented CLL-associated antigens represents a promising, low-side-effect therapeutic option to optimize treatment responses and eliminate residual tumor cells by inducing an anti-leukemic immune response. The iVAC-XS15-CLL01 trial is an open-label, first-in-human (FIH) Phase I trial, evaluating the CLL-VAC-XS15 vaccine in CLL patients undergoing BTKi-based therapy. The vaccine was developed from HLA-presented CLL-associated antigen peptides, identified through comparative mass-spectrometry-based immunopeptidome analyses of CLL versus healthy samples in a previous study. To facilitate rapid and cost-effective deployment, vaccine peptides are selected for each patient from a pre-manufactured "peptide warehouse" based on the patient's individual HLA allotype and CLL immunopeptidome. The trial enrolls 20 CLL patients, who receive up to three doses of the vaccine, adjuvanted with the toll-like-receptor (TLR) 1/2 ligand XS15 and emulsified in Montanide ISA 51 VG. The primary objective of the iVAC-XS15-CLL01 trial is to assess the safety and immunogenicity of the CLL-VAC-XS15 vaccine. Secondary objectives are to evaluate the vaccine impact on MRD, progression-free survival, and overall survival, as well as comprehensive immunophenotyping to characterize vaccine-induced T-cell responses. This Phase I trial aims to advance CLL treatment by enhancing immune-mediated disease clearance and guiding the design of subsequent Phase II/III trials to implement a new therapeutic strategy for CLL patients.

Peptide-based immunoprotection against Rhipicephalus microplus tick

The main agents for tick control are chemical acaricides. However, when used without technical guidance, they can lead to environmental damage and the development of resistant tick strains. In this context, vaccines are alternative o be used in integrated tick management format by combining with other effective tools. We isolated RNA from ticks Rhipicephalus microplus, prepared the library, and performed next-generation sequencing; a pipeline analysis was applied to identify the hypothetical proteins having immunogenic potential and their predicted immunogenic peptides. Twelve peptides, ranging from 12 to 38 amino acid residues, containing the selected epitopes from different targets were selected and synthesized in two forms: the pure peptide; and the peptide conjugated to keyhole limpet hemocyanin (KLH) carrier. These peptides were divided into two groups of six peptides each. The antigen formulations (groups 1 and 2) were prepared with conjugated peptides containing 200 µg of each peptide per dose emulsified with Montanide ISA 61VG (SEPPIC); the control treatment had the adjuvant formulation without peptides (group 3). To evaluate the protective efficacy, 15 weaned male calves (Angus breed) aged around 6 months to one year and weighing approximately 200–250 kg were divided into three groups of five animals each; they were immunized thrice, at an interval of 28 days. After immunization, all the calves infested with 15,000 larvae of Rhipicephalus microplus. Peptide epitopes were recognized by antibodies against host-specific IgGs using indirect ELISA. The mean of the antibody level was determined for each group and compared using analysis of variance with two factors (ANOVA). F-test was used to determine the significance of differences observed between the groups. The percentage efficacy was calculated based on the number of ticks, the weight of teleoginas, and the weight and hatchability of the eggs, compared to that in the control group. The evaluation of immunoprotection indicated efficacies of 69 and 51 %, respectively in Group 1 and 2.

Use of MONTANIDE ISA 78 VG as adjuvant for the manufacture of antibacterial vaccines for chickens

Relevance. The use of antimicrobial agents and vaccine prophylaxis are the main ways to prevent and control most diseases of bacterial etiology. At the same time, uncontrolled use of antimicrobial agents without determining the sensitivity of microorganisms, as a rule, does not give the opportunity to obtain a positive therapeutic result. At the same time, a competently designed scheme of inactivated vaccine administration, taking into account the epizootic situation, is an effective and safe tool for controlling bacterial diseases.In this regard, the results of testing of vaccine samples against bacterial diseases of birds based on the modernized oil adjuvant Montanide ISA 78 VG presented in this article are interesting and modern.Methods. Four vaccine samples were manufactured for research based on the oil adjuvant Montanide ISA 78 VG. The first sample of the vaccine is against avian salmonellosis, the second — against avian pasteurellosis, the third — against avian respiratory mycoplasmosis, the fourth — against avian hemophilosis. All manufactured vaccine samples were investigated for sterility, stability and viscosity of the emulsion according to generally accepted methods. To determine the reactogenicity and antigenic activity of the vaccine samples, young egg-laying hens aged 25–40 days were used.Results. Analysis of the obtained results of physical and biological properties of tested inactivated vaccines against bacterial diseases of birds allows us to conclude that the vaccine against respiratory mycoplasmosis of birds made on the basis of adjuvant Montanide ISA 78 VG is a safe and effective immunobiological preparation.

Safety and efficacy of C. muridarum vaccines adjuvanted with CpG-1826 and four concentrations of Montanide-ISA-720-VG

It is recommended that the adjuvant Montanide ISA 720 VG be used at a concentration of 70% v/v. At this concentration, Montanide causes at the site of immunization a local granuloma that can last for several weeks. To determine the safety and protective efficacy of a Chlamydia muridarum MOMP vaccine, formulated with CpG-1826 and four different concentrations of Montanide (70%, 50%, 30% and 10%), BALB/c (H-2d) female mice were immunized twice intramuscularly. Local reactogenicity was significant for vaccines formulated with 70% or 50% Montanide but not for those inoculated with 30% or 10% Montanide. Robust humoral and cell mediated memory immune responses were elicited by the 70%, 50% and 30% Montanide formulations. Mice were challenged intranasally with 104 C. muridarum inclusion forming units (IFU). Based on changes in body weight, lungs’s weight and number of IFU recovered, mice vaccinated with the 70%, 50% and 30% Montanide formulations were significantly protected, but not mice receiving 10% Montanide. To conclude, we recommend the 30% Montanide concentration to be tested in humans and animal models to determine its safety and efficacy, in comparison to the 70% Montanide concentration currently used. The 30% Montanide formulation could significantly facilitate licensing of this adjuvant for human use.

Evaluation in mice of cell-free produced CT584 as a Chlamydia vaccine antigen.

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and de-centralized production of recombinant protein vaccine antigens. Here, we use CFPS to produce the putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for use as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four-weeks later by an i.m. boost before respiratory challenge with 104 inclusion forming units (IFU) of Chlamydia muridarum. Immunization with CT584 generated robust antibody responses but weak cell mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lungs' weights and the presence of high numbers of IFUs in the lungs. While CT584 alone may not be the ideal vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens makes it an attractive technique for antigen production.

FusionVAC22_01: A phase I clinical trial evaluating a DNAJB1-PRKACA fusion transcript-based peptide vaccine combined with immune checkpoint inhibition for fibrolamellar hepatocellular carcinoma and other tumor entities carrying the oncogenic driver fusion.

TPS2702 Background: The DNAJB1-PRKACA fusion transcript is the driving force behind tumor development in fibrolamellar hepatocellular carcinoma (FL-HCC) and other tumor types, such as oncocytic neoplasms of the pancreas and bile ducts. Consequently, it serves as a versatile target for innovative cancer therapies. Recently, the DNAJB1-PRKACA fusion protein was recognized as a source of HLA-presented neoepitopes targetable by to T cell-based immunotherapy (1). The FusionVAC-22 neoepitope, derived from DNAJB1-PRKACA fusion, is computationally predicted to bind to 1,290 different HLA class II alleles. Furthermore, it includes embedded HLA class I ligands for 13 out of the 20 most common HLA class I alleles, covering 93.8% of the global population This broad coverage enables the widespread application of FusionVAC-22-based immunotherapies. The initial use of FusionVAC-22-based peptide vaccines, adjuvanted with the TLR1/2 agonist XS15 emulsified in Montanide ISA 51 VG, in two FL-HCC patients was well-tolerated and demonstrated the induction of robust and long-lasting T cell responses. Notably, T cell responses coincided with 32 months and 13 months of progression-free survival for the respective patients. Methods: Building on these promising outcomes, we have initiated a Phase I open-label, multicentric clinical trial to assess the immunogenicity, safety, toxicity, and initial signs of efficacy of the FusionVAC-22-based peptide vaccine combined with the immune checkpoint inhibitor (ICI) atezolizumab. The trial includes 20 patients with locally advanced or metastatic FL-HCC or other malignancies carrying the DNAJB1-PRKACA fusion transcript without available standard therapy. Key eligibility criteria involve the absence of autoimmune phenomena due to prior immunotherapy agents (≥ grade 3) and no history of tissue or organ allografts. The FusionVAC-22-based peptide vaccine is administered twice with a 4-week interval, with an option for a booster vaccination after 11 months. ICI administration begins on day 15 after the first vaccination and continues every 4 weeks for 1 year, followed by a 6-month follow-up. Primary trial objectives include assessing immunogenicity in terms of peptide-specific T cell responses up to 28 days after the second vaccination, as well as evaluating the safety and toxicity of the peptide vaccine in combination with ICI. Safety assessment is based on the frequency of adverse events according to CTCAE v5.0. Clinical efficacy is determined through iRECIST assessment on imaging. Additionally, disease control rate, quality of life, and overall and progression-free survival will be evaluated. So far, 3 out of 20 planned patients have been enrolled. 1. Bauer et al., Nat. Commun, 2022. Clinical trial information: NCT05937295 .

Developing a PmSLP3-based vaccine formulation that provides robust long-lasting protection against hemorrhagic septicemia-causing serogroup B and E strains of Pasteurella multocida in cattle.

Pasteurella multocida is a bacterial pathogen that causes a variety of infections across diverse animal species, with one of the most devastating associated diseases being hemorrhagic septicemia. Outbreaks of hemorrhagic septicemia in cattle and buffaloes are marked by rapid progression and high mortality. These infections have particularly harmful socio-economic impacts on small holder farmers in Africa and Asia who are heavily reliant on a small number of animals kept as a means of subsistence for milk and draft power purposes. A novel vaccine target, PmSLP-3, has been identified on the surface of hemorrhagic septicemia-associated strains of P. multocida and was previously shown to elicit robust protection in cattle against lethal challenge with a serogroup B strain. Here, we further investigate the protective efficacy of this surface lipoprotein, including evaluating the immunogenicity and protection upon formulation with a variety of adjuvants in both mice and cattle. PmSLP-3 formulated with Montanide ISA 61 elicited the highest level of serum and mucosal IgG, elicited long-lasting serum antibodies, and was fully protective against serogroup B challenge. Studies were then performed to identify the minimum number of doses required and the needed protein quantity to maintain protection. Duration studies were performed in cattle, demonstrating sustained serum IgG titres for 3 years after two doses of vaccine and full protection against lethal serogroup B challenge at 7 months after a single vaccine dose. Finally, a serogroup E challenge study was performed, demonstrating that PmSLP-3 vaccine can provide protection against challenge by the two serogroups responsible for hemorrhagic septicemia. Together, these data indicate that PmSLP-3 formulated with Montanide ISA 61 is an immunogenic and protective vaccine against hemorrhagic septicemia-causing P. multocida strains in cattle.

Development of novel Streptococcus equi vaccines with an assessment of their immunizing potentials and protective efficacies

Strangles is a highly contagious disease of the equine upper respiratory tract caused by Streptococcus equi subspecies. Streptococcus equi subsp. equi (S. equi) and Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) was isolated, as local, hot, and field strains, from horses clinically suffering from respiratory distress. The isolated Streptococci were identified using bacteriological and molecular techniques. Four formulations of inactivated S. equi vaccines were developed and evaluated. The first formulation was prepared using the S. equi isolates, adjuvanted with MONTANIDE GEL adjuvant, while the second formulation was adjuvanted with MONTANIDE ISA-70 adjuvant. The other 2 formulations were inactivated combined vaccines prepared from both S. equi and S. zooepidemicus isolates. The 3rd formulation was the combined isolates adjuvanted with MONTANIDE GEL while the 4th formulation was the combined isolates adjuvanted with MONTANIDE ISA-70. The developed vaccines’ physical properties, purity, sterility, safety, and potency were ensured. The immunizing efficacy was determined in isogenic BALB/c mice and white New Zealand rabbits using the passive hemagglutination test. Also, the antibodies’ titer of the combined S. equi and S. zooepidemicus vaccine adjuvanted with MONTANIDE ISA-70 in foals was tracked using an indirect enzyme-linked immunosorbent assay. The protective efficacy of the developed vaccines was determined using a challenge test in both laboratory and field animal models, where a 75% protection rate was achieved. The combined vaccine proved to be more efficacious than the monovalent vaccine. Also, the MONTANIDE ISA-70 adjuvant provided significant protective efficacy than the MONTANIDE GEL. The current work is introducing a very promising mitigative and strategic controlling solution for strangles.

Abstract PO2-17-12: Phase I Trial of alpha-lactalbumin vaccine in high risk operable triple negative breast cancer (TNBC) and patients at high genetic risk for TNBC

Abstract Background: Triple-negative breast cancer (TNBC) has a poor prognosis and may be associated with germline mutations. α-Lactalbumin (aLA) is expressed in lactating breasts but not at other times or in other tissues. Expression of aLA is found in 70% of TNBC (PMID: 27322324) so could be an immunologic target for TNBC based on the “retired protein hypothesis” (PMID: 31926646). In pre-clinical studies, vaccination with aLA provided protection from development of autochthonous tumors in transgenic murine models of breast cancer and inhibited growth of established 4T1 transplantable breast tumors in BALB/c mice (PMID: 20512124). Methods: To determine the safety and immunogenicity of aLA, patients with early stage TNBC are being entered in a Phase I trial of aLA with GMP-grade zymosan adjuvant in Montanide ISA 51 VG vehicle. Subjects receive 3 vaccinations given once every 2 weeks. Events of Common Terminology Criteria for Adverse Events (CTCAE) grade ≥ 2 are considered dose-limiting toxicities (DLTs). Results: CTCAE toxicity by dose level is summarized below. All DLTs were injection site reactions, with ulceration and need for incisional drainage representing the grade 3 events. Seven (7) of 10 patients assayed to date have met protocol specified definitions of an immune response based on ELISpot assays to determine frequencies of T cells producing IFNγ and IL-17 in response to recombinant aLA. Assays for an additional 6 patients will be available in September 2023. Conclusion: Dose level ≤ 2 appears to be the maximum tolerated dose. Based on immune response, additional intermediate dose levels may be studied. An additional cohort of patients receiving concurrent anti-PD1 treatment is being accrued. Accrual of patients with BRCA1/2 or PALB2 mutations planning to undergo prophylactic mastectomy is beginning in order to define the toxicity and immunologic effects in this group and to determine whether inflammatory changes from occult lactational foci will be produced. Funding Source: Department of Defense (W81XWH-17-1-0592 and W81XWH-17-1-0593) Table 1 Worst Toxicity by Dose Level Citation Format: Justin Johnson, Emily Rhoades, Holly Levengood, Halle Moore, Megan Kruse, Erin Roesch, Jame Abraham, Brenna Elliott, Rachel Swartz, Holly Pederson, Elena Haury, Azka Ali, Tiffany Onger, Andrew Sciallis, Zahraa Al-Hilli, Wei Wei, Thaddeus Stappenbeck, George Budd. Phase I Trial of alpha-lactalbumin vaccine in high risk operable triple negative breast cancer (TNBC) and patients at high genetic risk for TNBC [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-17-12.

Adjuvant screening of the Senecavirus A inactivated vaccine in mice and evaluation of its immunogenicity in pigs

BackgroundSenecavirus A (SVA) causes an emerging vesicular disease (VD) with clinical symptoms indistinguishable from other vesicular diseases, including vesicular stomatitis (VS), foot-and-mouth disease (FMD), and swine vesicular disease (SVD). Currently, SVA outbreaks have been reported in Canada, the U.S.A, Brazil, Thailand, Vietnam, Colombia, and China. Based on the experience of prevention and control of FMDV, vaccines are the best means to prevent SVA transmission.ResultsAfter preparing an SVA inactivated vaccine (CH-GX-01-2019), we evaluated the immunogenicity of the SVA inactivated vaccine mixed with Imject® Alum (SVA + AL) or Montanide ISA 201 (SVA + 201) adjuvant in mice, as well as the immunogenicity of the SVA inactivated vaccine combined with Montanide ISA 201 adjuvant in post-weaned pigs. The results of the mouse experiment showed that the immune effects in the SVA + 201 group were superior to that in the SVA + AL group. Results from pigs immunized with SVA inactivated vaccine combined with Montanide ISA 201 showed that the immune effects were largely consistent between the SVA-H group (200 µg) and SVA-L group (50 µg); the viral load in tissues and blood was significantly reduced and no clinical symptoms occurred in the vaccinated pigs.ConclusionsMontanide ISA 201 is a better adjuvant choice than the Imject® Alum adjuvant in the SVA inactivated vaccine preparation, and the CH-GX-01-2019 SVA inactivated vaccine can provide effective protection for pigs.

FusionVAC22_01: A phase I clinical trial evaluating a DNAJB1-PRKACA fusion transcript-based peptide vaccine combined with immune checkpoint inhibition for fibrolamellar hepatocellular carcinoma and other tumor entities carrying the oncogenic driver fusion.

TPS580 Background: The DNAJB1-PRKACA fusion transcript is the driver of tumor pathogenesis in fibrolamellar hepatocellular carcinoma (FL-HCC) as well as in other tumor entities, e.g. oncocytic neoplasms of the pancreas and bile duct, thus representing a broad target for novel therapies in cancer. Recently, the DNAJB1-PRKACA fusion protein was identified as a source for HLA-presented neoepitopes that can be targeted by T-cell-based immunotherapy (Bauer et al. Nat. Commun., 2022). The DNAJB1-PRKACA fusion-derived neoepitope FusionVAC-22 is in silico predicted to bind to 1,290 HLA class II alleles and contains HLA class I ligands from 13 of the 20 most frequent HLA class I alleles that cover 93.8% of the world population with at least one HLA allotype, enabling the broad application of FusionVAC-22-based immunotherapies. The first application of FusionVAC-22-based peptide vaccines adjuvanted with the TLR1/2 agonist XS15 emulsified in Montanide ISA 51 VG in two FL-HCC patients was well tolerated and showed the induction of profound and long-lasting T-cell responses. Of note, T-cell responses were accompanied by progression-free survival of both patients for so far 32 months and 13 months, respectively. Methods: Based on these encouraging results, we established a Phase I open-label, multicentric clinical trial to evaluate the immunogenicity along with safety and toxicity, as well as first signs of efficacy of the FusionVAC-22 based peptide vaccine, combined with the immune checkpoint inhibitor (ICI) atezolizumab, in 20 patients with locally advanced or metastatic FL-HCC or other malignant diseases that carry the DNAJB1-PRKACA fusion transcript. Further, key eligibility criteria include the absence of autoimmune phenomena due to prior immunotherapy agents (≥ grade 3) as well as tissue or organ allografts. The FusionVAC-22-based peptide vaccine is applied twice in a 4-weekly interval with the opportunity of a booster vaccination after 11 months. Atezolizumab application starts on day 15 after first vaccination and is continued 4-weekly for 1 year, followed by a 6-month follow-up. The primary objectives of the trial are immunogenicity, in terms of induction of peptide specific T-cell responses until 28 days after second vaccination, as well as safety and toxicity of the peptide vaccine in combination with ICI. Safety assessment is based on the frequency of adverse events according to CTCAE v5.0. Clinical efficacy is determined by iRECIST assessment on imaging. Furthermore, disease control rate, quality of life as well as overall and progression free survival will be assessed. Clinical trial information: NCT05937295 .

Protective Efficacy of a Locally Prepared Bivalent Duck Viral Hepatitis “Serotypes 1 and 3” Inactivated Vaccine

Abstract The spread of duck hepatitis virus (DHV) in Egypt with its’ most prevalent genotypes (1 and 3) causes vast economic losses in the duck industry despite the regular vaccination with live attenuated vaccines. However, the use of live-vaccines is considered a potential risk for the non-vaccinated birds due to the viral shedding from vaccinated populations. The current study aimed to evaluate the protective efficacy and safety of two inactivated monovalent and one bivalent vaccines against DHV 1 and 3 genotypes. The inactivated monovalent (DHAV-1, DHAV-3) and bivalent (DHAV-1+3) vaccines were produced by using Montanide ISA 70 oil as an adjuvant. Three groups of 4-week-old ducklings (n=50) were vaccinated with one of the three vaccines, respectively. One group of ducklings was used as negative control (n=25). The immune-response of the vaccinated groups was measured by Virus Neutralization Test and expressed with Neutralizing Indices (NIs). The NIs for the bivalent vaccine group (5.6 and 5.4) were higher compared to the monovalent vaccine groups (5.0 and 4.7). In conclusion, the use of inactivated bivalent DHV vaccine could produce higher protective efficacy compared to the monovalent DHAV-1 and DHAV-3 vaccines.

Compared to Aluminum Hydroxide Adjuvant, Montanide ISA 206 VG Induces a Higher and More Durable Neutralizing Antibody Response against FMDV in Goats.

Foot-and-mouth disease (FMD) has a high prevalence in cloven-hoofed animals. It is also highly contagious and remains a serious threat to livestock worldwide. Despite the widespread vaccination program in Iran, outbreaks of FMD continue to occur. Vaccination is one of the most effective methods of preventing FMD. The vaccines used in Iran are of the inactivated type and contain several serotypes. Since inactivated vaccines without adjuvants do not induce a high and durable antibody response, it is necessary to use adjuvants. Montanide ISA 206 VG is a mineral oil-based adjuvant that produces a water-in-oil-in-water (w:o:w) emulsion in vaccine preparations. However, a large number of manufacturers in Iran and around the world still use alum adjuvant (with or without saponin) to produce the FMD vaccine. This study used Montanide ISA 206 and alum adjuvants to administer the O2010 serotype of the FMD virus to goats. A total of six goats were divided randomly into three groups. Vaccines were administered subcutaneously twice, at a one-month interval. Blood sampling was done at different times, and the micro-neutralization method was used to measure the neutralizing antibody titer in each serum. Seven days after the second vaccination, the alum group's antibody titer was higher but not statistically significant. However, from the 28th day after the second injection until the end of the study, the Montanide ISA 206 group's antibody titer was significantly higher than that of the alum group. Six months after the second injection, the antibody titer in the ISA 206 group remained at the peak level, while in the alum group, it decreased and reached the minimum protective level. Nine months after the second injection, the antibody titer remained at its peak level in the ISA 206 group, whereas it dropped significantly in the alum group. Based on the findings, ISA 206 VG is capable of generating long-term humoral immunity in goats against the FMD serotype O2010 and could replace aluminum hydroxide adjuvants in FMD vaccine preparations.