Use of a Clostridioides difficile Murine Immunization and Challenge Model to Evaluate Single and Combination Vaccine Adjuvants Consisting of Alum and NKT Cell-Activating Ligands

Adjuvant combinations may enhance or broaden the expression of immune responses to vaccine antigens. Information on whether established Alum type adjuvants can be combined with experimental CD1d ligand adjuvants is currently lacking. In this study, we used a murine Clostridioides difficile immunization and challenge model to evaluate Alum (Alhydrogel™), α-galactosylceramide (α-GC), and one of its analogs 7DW8-5 singly and in combination as vaccine adjuvants. We observed that the Alum/α-GC combination caused modest enhancement of vaccine antigen-specific IgG1 and IgG2b responses, and a broadening to include IgG2c that did not significantly impact overall protection. Similar observations were made using the Alum/7DW8-5 combination. Examination of the impact of adjuvants on NKT cells revealed expansion of invariant NKT (iNKT) cells with modest expansion of their iNKTfh subset and little effect on diverse NKT (dNKT) cells. Side effects of the adjuvants was determined and revealed transient hepatotoxicity when Alum/α-GC was used in combination but not singly. In summary these results showed that the Alum/α-GC or the Alum/7DW8-5 combination could exert distinct effects on the NKT cell compartment and on isotype switch to produce Th1-driven IgG subclasses in addition to Alum/Th2-driven subclasses. While Alum alone was efficacious in stimulating IgG-mediated protection, and α-GC offered no apparent additional benefit in the C. difficile challenge model, the work herein reveals immune response features that could be optimized and harnessed in other vaccine contexts.

Elucidating functional epitopes within the N-terminal region of malaria transmission blocking vaccine antigen Pfs230

Pfs230 is a leading malaria transmission blocking vaccine (TBV) candidate. Comprising 3135 amino acids (aa), the large size of Pfs230 necessitates the use of sub-fragments as vaccine immunogens. Therefore, determination of which regions induce functional antibody responses is essential. We previously reported that of 27 sub-fragments spanning the entire molecule, only five induced functional antibodies. A “functional” antibody is defined herein as one that inhibits Plasmodium falciparum parasite development in mosquitoes in a standard membrane-feeding assay (SMFA). These five sub-fragments were found within the aa 443–1274 range, and all contained aa 543–730. Here, we further pinpoint the location of epitopes within Pfs230 that are recognized by functional antibodies using antibody depletion and enrichment techniques. Functional epitopes were not found within the aa 918–1274 region. Within aa 443–917, further analysis showed the existence of functional epitopes not only within the aa 543–730 region but also outside of it. Affinity-purified antibodies using a synthetic peptide matching aa 543–588 showed activity in the SMFA. Immunization with a synthetic peptide comprising this segment, formulated either as a carrier-protein conjugate vaccine or with a liposomal vaccine adjuvant system, induced antibodies in mice that were functional in the SMFA. These findings provide key insights for Pfs230-based vaccine design and establish the feasibility for the use of synthetic peptide antigens for a malaria TBV.

Immunization Trials with Recombinant Major Sperm Protein of the Bovine Lungworm Dictyocaulus viviparus

The lungworm Dictyocaulus viviparus is one of the most economically important bovine parasites in temperate climate regions. Following infection, D. viviparus induces a temporary protective immunity, and a vaccine based on attenuated, infective larvae is commercially available. However, due to several disadvantages of the live vaccine, the development of a recombinant subunit vaccine is highly desirable. Therefore, the major sperm protein (MSP), which is essential for the parasite’s reproduction, was tested as a recombinantly Escherichia coli-expressed glutathione-S-transferase (GST)-fused vaccine antigen in immunization trials with two different adjuvants, Quil A and Al(OH)3. Calves (N = 4 per group) were immunized on study day (SD) 0, 21 and 42 and given a challenge infection on SD 63–65. The two control groups received only the respective adjuvant. Based on geometric means (GM), a 53.64% reduction in larvae per female worm was observed in the rMSP Quil A group vs. its control group (arithmetic means (AM): 54.43%), but this difference was not statistically significant. In the rMSP Al(OH)3 group, the mean number of larvae per female worm was even higher than in the respective control group (GM: 9.24%, AM: 14.14%). Furthermore, male and female worm burdens and the absolute number of larvae did not differ significantly, while the Al(OH)3 control group harbored significantly longer worms than the vaccinated group. Vaccinated animals showed a rise in rMSP-specific antibodies, particularly IgG and its subclass IgG1, and the native protein was detected by immunoblots. Although rMSP alone did not lead to significantly reduced worm fecundity, it might still prove useful as part of a multi-component vaccine.

S-540956, a CpG Oligonucleotide Annealed to a Complementary Strand With an Amphiphilic Chain Unit, Acts as a Potent Cancer Vaccine Adjuvant by Targeting Draining Lymph Nodes

Robust induction of cancer-antigen-specific CD8+ T cells is essential for the success of cancer peptide vaccines, which are composed of a peptide derived from a cancer-specific antigen and an immune-potentiating adjuvant, such as a Toll-like receptor (TLR) agonist. Efficient delivery of a vaccine antigen and an adjuvant to antigen-presenting cells in the draining lymph nodes (LNs) holds key to maximize vaccine efficacy. Here, we developed S-540956, a novel TLR9-agonistic adjuvant consisting of B-type CpG ODN2006 (also known as CpG7909), annealed to its complementary sequence oligodeoxynucleotide (ODN) conjugated to a lipid; it could target both a cancer peptide antigen and a CpG-adjuvant in the draining LNs. S-540956 accumulation in the draining LNs and activation of plasmacytoid dendritic cells (pDCs) were significantly higher than that of ODN2006. Mechanistic analysis revealed that S-540956 enhanced the induction of MHC class I peptide-specific CD8+ T cell responses via TLR9 in a CD4+ T cell-independent manner. In mice, the therapeutic effect of S-540956-adjuvanted with a human papillomavirus (HPV)-E7 peptide vaccine against HPV-E7-expressing TC-1 tumors was significantly better than that of an ODN2006-adjuvanted vaccine. Our findings demonstrate a novel adjuvant discovery with the complementary strand conjugated to a lipid, which enabled draining LN targeting and increased ODN2006 accumulation in draining LNs, thereby enhancing the adjuvant effect. Our findings imply that S-540956 is a promising adjuvant for cancer peptide vaccines and has a high potential for applications in various vaccines, including recombinant protein vaccines.

Hypersecretion of vaccine antigen outer membrane lipoprotein A in Corynebacterium glutamicum through high-throughput based development process

Outer membrane lipoprotein A (OmlA) is a vaccine antigen against porcine contagious pleuropneumonia (PCP), a disease severely affecting the swine industry. Here, we aimed to systematically potentiate the secretory production of OmlA in Corynebacterium glutamicum (C. glutamicum), a widely used microorganism in the food industry, by establishing a holistic development process based on our high-throughput culture platform. The expression patterns, expression element combinations, medium composition, and induction conditions were comprehensively screened or optimized in microwell plates (MWPs), followed by fermentation parameter optimization in a 4×1 L parallel fermentation system (CUBER4). An unprecedented yield of 1.01 g/L OmlA was ultimately achieved in a 5-L bioreactor following the scaling-up strategy of fixed oxygen mass transfer coefficient (kLa), and the produced OmlA antigen showed well-protective immunity against Actinobacillus pleuropneumoniae challenge. This result provides a rapid and reliable pipeline to achieve the hyper-production of OmlA, and possibly other recombinant vaccines, in C. glutamicum.

Innovations in the Insect Cell Expression System for Industrial Recombinant Vaccine Antigen Production

The insect cell expression system has previously been proposed as the preferred biosecurity strategy for production of any vaccine, particularly for future influenza pandemic vaccines. The development and regulatory risk for new vaccine candidates is shortened as the platform is already in use for the manufacturing of the FDA-licensed seasonal recombinant influenza vaccine Flublok®. Large-scale production capacity is in place and could be used to produce other antigens as well. However, as demonstrated by the 2019 SARS-CoV-2 pandemic the insect cell expression system has limitations that need to be addressed to ensure that recombinant antigens will indeed play a role in combating future pandemics. The greatest challenge may be the ability to produce an adequate quantity of purified antigen in an accelerated manner. This review summarizes recent innovations in technology areas important for enhancing recombinant-protein production levels and shortening development timelines. Opportunities for increasing product concentrations through vector development, cell line engineering, or bioprocessing and for shortening timelines through standardization of manufacturing processes will be presented.

Glycan Masking of Epitopes in the NTD and RBD of the Spike Protein Elicits Broadly Neutralizing Antibodies Against SARS-CoV-2 Variants

Glycan-masking the vaccine antigen by mutating the undesired antigenic sites with an additional N-linked glycosylation motif can refocus B-cell responses to desired epitopes, without affecting the antigen’s overall-folded structure. This study examined the impact of glycan-masking mutants of the N-terminal domain (NTD) and receptor-binding domain (RBD) of SARS-CoV-2, and found that the antigenic design of the S protein increases the neutralizing antibody titers against the Wuhan-Hu-1 ancestral strain and the recently emerged SARS-CoV-2 variants Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Our results demonstrated that the use of glycan-masking Ad-S-R158N/Y160T in the NTD elicited a 2.8-fold, 6.5-fold, and 4.6-fold increase in the IC-50 NT titer against the Alpha (B.1.1.7), Beta (B.1.351) and Delta (B.1.617.2) variants, respectively. Glycan-masking of Ad-S-D428N in the RBD resulted in a 3.0-fold and 2.0-fold increase in the IC-50 neutralization titer against the Alpha (B.1.1.7) and Beta (B.1.351) variants, respectively. The use of glycan-masking in Ad-S-R158N/Y160T and Ad-S-D428N antigen design may help develop universal COVID-19 vaccines against current and future emerging SARS-CoV-2 variants.

Molecular Characterization of a Tetraspanin TSP11 Gene in Echinococcus granulosus and Evaluation Its Immunoprotection in Model Dogs

Cystic echinococcosis (CE) is a cosmopolitan zoonosis caused by the larval stage of Echinococcus granulosus, which affects humans and a wide range of mammalian intermediate hosts. Parasite tetraspanin proteins are crucial for host-parasite interactions, and therefore they may be useful for vaccine development or disease diagnosis. In the present study, the major antigen coding sequence of tetraspanin 11 (Eg-TSP11) from E. granulosus was determined. The results of immunolocalization showed that Eg-TSP11 was mainly located in the tegument of adult worms and protoscoleces. Western blotting analysis showed that the serum from dogs injected with recombinant Eg-TSP11 (rEg-TSP11) could recognize Eg-TSP11 among natural protoscolex proteins. Moreover, the serum from dogs with E. granulosus infection also recognized rEg-TSP11. Serum indirect enzyme-linked immunosorbent assays demonstrated that IgG levels gradually increased after the first immunization with rEg-TSP11 compared with those in the control group. Furthermore, the serum levels of interleukin 4, interleukin 5, and interferon gamma were significantly altered in the rEg-TSP11 group. Importantly, we found that vaccination with rEg-TSP11 significantly decreased worm burden and inhibited segment development in a dog model of E. granulosus infection. Based on these findings, we speculated that rEg-TSP11 might be a potential candidate vaccine antigen against E. granulosus infection in dogs.