Developing a Stabilizing Formulation of a Live Chimeric Dengue Virus Vaccine Dry Coated on a High-Density Microarray Patch

Alternative delivery systems such as the high-density microarray patch (HD-MAP) are being widely explored due to the variety of benefits they offer over traditional vaccine delivery methods. As vaccines are dry coated onto the HD-MAP, there is a need to ensure the stability of the vaccine in a solid state upon dry down. Other challenges faced are the structural stability during storage as a dried vaccine and during reconstitution upon application into the skin. Using a novel live chimeric virus vaccine candidate, BinJ/DENV2-prME, we explored a panel of pharmaceutical excipients to mitigate vaccine loss during the drying and storage process. This screening identified human serum albumin (HSA) as the lead stabilizing excipient. When bDENV2-coated HD-MAPs were stored at 4 °C for a month, we found complete retention of vaccine potency as assessed by the generation of potent virus-neutralizing antibody responses in mice. We also demonstrated that HD-MAP wear time did not influence vaccine deposition into the skin or the corresponding immunological outcomes. The final candidate formulation with HSA maintained ~100% percentage recovery after 6 months of storage at 4 °C.

IMMU-04. VACCINATING AGAINST NOVEL CYTOMEGALOVIRUS ANTIGENS IN GLIOBLASTOMA USING A PEPTIDE VACCINE IN COMBINATION WITH TEMOZOLOMIDE

INTRODUCTION Cytomegalovirus (CMV) antigens are excellent anti-tumor immunotherapeutic targets in glioblastoma (GBM). The PERFORMANCE trial (IRB-pro34208, IND-15846) assessed the feasibility, safety and optimal adjuvant temozolomide (TMZ) regimen to be used with PEP-CMV vaccination in adults with newly-diagnosed GBM. METHODS Seropositive CMV patients (n=16) were randomized into two arms and treated with standard of care RT-TMZ (SOC) (150-200 mg/m2/day on days 1-5 per 28-day cycle) or dose-intensive TMZ (DI) (75-100 mg/m2/day on days 1-21 per 28-day cycle). Patients received intradermal PEP-CMV vaccines (500μg of CMVpp65 synthetic long peptide (SLP) mixed with Montanide ISA-51) on days 23, 37 and 51 following TMZ. All patients received tetanus/diphtheria toxoid (Td) preconditioning at the vaccination site on day 22. Serum cytokine levels were measured pre-vaccination, 1-hour and 2-hours post vaccination. PEP-CMV specific circulating PBMCs were quantified at baseline and after each vaccine. RESULTS Of the 16 trial patients, 7 experienced site-reactions, 4 had grade-II Immune Related Adverse Events (IRAEs), and 4 experienced flu-like grade-III IRAEs. Inflammatory cytokines (IL-2, IFNγ, MIP-1a, IL-8, TNFα, and IL-10) were elevated in patients with grade-III responses 2-hours post vaccine 1. Td p2/p30 specific PBMC levels were similar between IRAEs. However, pp65 responsive PBMCs were elevated at baseline in patients with grade-III reactions compared to site-reaction suggesting pre-existing peptide specific responses may lead to increased vaccine immunogenicity. PBMCs specific for pp65 increased with number of consecutive vaccines. No difference in progression free survival (PFS) or overall survival (OS) was observed between TMZ regimens. CONCLUSION PEP-CMV vaccination with Td preconditioning is feasible and generates immune responses specific to pp65 in patients with newly diagnosed GBM. Importantly, IRAEs were associated with antitumor efficacy. The mild IRAEs in PERFORMANCE are likely indicative of vaccine potency and can be managed through standard premedication as has been used in other trials with similar IRAEs.

Epitope order Matters in multi-epitope-based peptide (MEBP) vaccine design: An in silico study

With different countries facing multiple waves, with some SARS-CoV-2 variants more deadly and virulent, the COVID-19 pandemic is becoming more dangerous by the day and the world is facing an even more dreadful extended pandemic with exponential positive cases and increasing death rates. There is an urgent need for more efficient and faster methods of vaccine development against SARS-CoV-2. Compared to experimental protocols, the opportunities to innovate are very high in immunoinformatics/in silico approaches especially with the recent adoption of structural bioinformatics in peptide vaccine design. In recent times, multi-epitope-based peptide vaccine candidates (MEBPVCs) have shown extraordinarily high humoral and cellular responses to immunization. Most of the publications claim that respective reported MEBPVC(s) assembled using a set of in silico predicted epitopes, to be the computationally validated potent vaccine candidate(s) ready for experimental validation. However, in this article, for a given set of predicted epitopes, it is shown that the published MEBPVC is one among the many possible variants and there is high likelihood of finding more potent MEBPVCs than the published candidate. To test the same, a methodology is developed where novel MEBP variants are derived by changing the epitope order of the published MEBPVC. Further, to overcome the limitations of current qualitative methods of assessment of MEBPVC, to enable quantitative comparison, ranking, and the discovery of more potent MEBPVCs, novel predictors, Percent Epitope Accessibility (PEA), Receptor specific MEBP vaccine potency(RMVP), MEBP vaccine potency(MVP) are introduced. The MEBP variants indeed showed varied MVP scores indicating varied immunogenicity. When the MEBP variants were ranked in descending order of their MVP scores, the published MEBPVC had the least MVP score. Further, the MEBP variants with IDs, SPVC_387 and SPVC_206, had the highest MVP scores indicating these variants to be more potent MEBPVCs than the published MEBPVC and hence should be prioritized for experimental testing and validation. Through this method, more vaccine candidates will be available for experimental validation and testing. This study also opens the opportunity to develop new software tools for designing more potent MEBPVCs in less time. The computationally validated top-ranked MEBPVCs must be experimentally tested, validated, and verified. The differences and deviations between experimental results and computational predictions provide an opportunity for improving and developing more efficient algorithms and reliable scoring schemes and software.

Novel Capsid-Specific Single-Domain Antibodies with Broad Foot-and-Mouth Disease Strain Recognition Reveal Differences in Antigenicity of Virions, Empty Capsids, and Virus-Like Particles

Foot-and-mouth disease (FMD) vaccine efficacy is mainly determined by the content of intact virions (146S) and empty capsids (75S). Both particles may dissociate into 12S subunits upon vaccine manufacturing, formulation, and storage, reducing vaccine potency. We report the isolation of capsid-specific llama single-domain antibodies (VHHs) with broad strain recognition that can be used to quantify intact capsids in FMD vaccines by double antibody sandwich (DAS) ELISA. One capsid-specific VHH displayed remarkably broad strain reactivity, recognizing 14 strains representing the 13 most important lineages of serotype A, and two VHHs cross-reacted with other serotypes. We additionally show that the newly isolated VHHs, as well as previously characterized VHHs, can be used to identify antigenic differences between authentic 146S and 75S capsids, as well as corresponding genetically engineered virus-like particles (VLPs). Our work underscores that VHHs are excellent tools for monitoring the quantity and stability of intact capsids during vaccine manufacturing, formulation, and storage, and additionally shows that VHHs can be used to predict the native-like structure of VLPs.

Broad Reactivity Single Domain Antibodies against Influenza Virus and Their Applications to Vaccine Potency Testing and Immunotherapy

The antigenic variability of influenza presents many challenges to the development of vaccines and immunotherapeutics. However, it is apparent that there are epitopes on the virus that have evolved to remain largely constant due to their functional importance. These more conserved regions are often hidden and difficult to access by the human immune system but recent efforts have shown that these may be the Achilles heel of the virus through development and delivery of appropriate biological drugs. Amongst these, single domain antibodies (sdAbs) are equipped to target these vulnerabilities of the influenza virus due to their preference for concave epitopes on protein surfaces, their small size, flexible reformatting and high stability. Single domain antibodies are well placed to provide a new generation of robust analytical reagents and therapeutics to support the constant efforts to keep influenza in check.