Carrier diversity and chemical ligations in the toolbox for designing tumor-associated carbohydrate antigens (TACAs) as synthetic vaccine candidates.

This review highlights the recent development in the use of carriers of increasing simplicities and versatile chemical ligation processes leading to synthetic vaccine candidates against tumor-associated carbohydrate antigens (TACAs). After briefly covering their structures, functions, occurrence, and biosynthesis, an overview of common conjugation chemistry is described with an emphasis on the versatile alkenyl glycosides as starting materials toward glycoconjugate syntheses. This is followed by a successive description of the numerous scaffolds and carriers used to progressively improve and simplify glycovaccine formulations. Throughout a systematic investigation of the various architectures involved, a critical description of the basic principles discovered en route to effective immune responses is disclosed wherein it is found that size, shape, densities, and carriers are all key factors involved towards successful vaccines.

Streamlining Peptide Mapping LC-MS Approach for Studying Fusion Peptide-Conjugated Vaccine Immunogens.

A newly introduced HIV-1 vaccination utilizes a fusion peptide (FP)-based immunogen-carrier conjugate system, where the FP is coupled to a protein carrier via a bifunctional linker. Such heterogeneous materials present a challenge for the routine product quality assessment. Peptide mapping LC-MS analysis has become an indispensable tool for assessing the site-specific conjugation ratio, estimating site occupancy, monitoring conjugation profiles, and analyzing post-translational modifications (PTMs) and disulfide bonds as well as high-order protein structures. To streamline the peptide mapping approach to match the needs of a fast-paced conjugate vaccine product characterization, a selection of signature fragment ions generated by MSE fragmentation was successfully applied to assess the product quality at the different stages of a conjugates' manufacturing process with an emphasis on monitoring the amount of a reactive linker. This technique was employed in different conjugation studies of the protein carriers, linkers, and FP compositions as well as the cross-linked species formed during stress-degradation studies. Multiple derivatives of the intermediate and final conjugated products formed during a multistaged synthesis were monitored by means of the sensitive extracted-ion chromatogram (XIC) profiling and were included in the estimation of the site-specific conjugation loads. Differentiation of the conjugates with various FP compositions was demonstrated. The conjugation site occupancy was evaluated with respect to the solvent exposure of Lys residues. The findings of these LC-MS studies greatly aided in choosing the best conjugation strategy to ensure that the final recombinant tetanus toxoid heavy chain (rTTHc) product is chemically inert and represents a safe vaccine candidate for clinical evaluation.

Site-Specific and Stable Conjugation of the SARS-CoV-2 Receptor-Binding Domain to Liposomes in the Absence of Any Other Adjuvants Elicits Potent Neutralizing Antibodies in BALB/c Mice.

Understanding immune responses toward viral infection will be useful for potential therapeutic intervention and offer insights into the design of prophylactic vaccines. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. To understand the complex immune responses toward SARS-CoV-2 infection, here we developed a method to express and purify the recombinant and engineered viral receptor-binding domain (RBD) to more than 95% purity. We could encapsulate RNA molecules into the interior of a virion-sized liposome. We conjugated the purified RBD proteins onto the surface of the liposome in an orientation-specific manner with defined spatial densities. Both the encapsulation of RNAs and the chemical conjugation of the RBD protein on liposome surfaces were stable under physiologically relevant conditions. In contrast to soluble RBD proteins, a single injection of RBD-conjugated liposomes alone, in the absence of any other adjuvants, elicited RBD-specific B cell responses in BALB/c mice, and the resulting animal sera could potently neutralize HIV-1 pseudovirions that displayed the SARS-CoV-2 spike proteins. These results validate these supramolecular structures as a novel and effective tool to mimic the structure of enveloped viruses, the use of which will allow systematic dissection of the complex B cell responses to SARS-CoV-2 infection.

Targeted Delivery of mRNA with One-Component Ionizable Amphiphilic Janus Dendrimers.

Targeted and efficient delivery of nucleic acids with viral and synthetic vectors is the key step of genetic nanomedicine. The four-component lipid nanoparticle synthetic delivery systems consisting of ionizable lipids, phospholipids, cholesterol, and a PEG-conjugated lipid, assembled by microfluidic or T-tube technology, have been extraordinarily successful for delivery of mRNA to provide Covid-19 vaccines. Recently, we reported a one-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) synthetic delivery system for mRNA relying on amphiphilic Janus dendrimers and glycodendrimers developed in our laboratory. Amphiphilic Janus dendrimers consist of functional hydrophilic dendrons conjugated to hydrophobic dendrons. Co-assembly of IAJDs with mRNA into dendrimersome nanoparticles (DNPs) occurs by simple injection in acetate buffer, rather than by microfluidic devices, and provides a very efficient system for delivery of mRNA to lung. Here we report the replacement of most of the hydrophilic fragment of the dendron from IAJDs, maintaining only its ionizable amine, while changing its interconnecting group to the hydrophobic dendron from amide to ester. The resulting IAJDs demonstrated that protonated ionizable amines play dual roles of hydrophilic fragment and binding ligand for mRNA, changing delivery from lung to spleen and/or liver. Replacing the interconnecting ester with the amide switched the delivery back to lung. Delivery predominantly to liver is favored by pairs of odd and even alkyl groups in the hydrophobic dendron. This simple structural change transformed the targeted delivery of mRNA mediated with IAJDs, from lung to liver and spleen, and expands the utility of DNPs from therapeutics to vaccines.

The self-assembled nanoparticle-based trimeric RBD mRNA vaccine elicits robust and durable protective immunity against SARS-CoV-2 in mice.

As COVID-19 continues to spread rapidly worldwide and variants continue to emerge, the development and deployment of safe and effective vaccines are urgently needed. Here, we developed an mRNA vaccine based on the trimeric receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein fused to ferritin-formed nanoparticles (TF-RBD). Compared to the trimeric form of the RBD mRNA vaccine (T-RBD), TF-RBD delivered intramuscularly elicited robust and durable humoral immunity as well as a Th1-biased cellular response. After further challenge with live SARS-CoV-2, immunization with a two-shot low-dose regimen of TF-RBD provided adequate protection in hACE2-transduced mice. In addition, the mRNA template of TF-RBD was easily and quickly engineered into a variant vaccine to address SARS-CoV-2 mutations. The TF-RBD multivalent vaccine produced broad-spectrum neutralizing antibodies against Alpha (B.1.1.7) and Beta (B.1.351) variants. This mRNA vaccine based on the encoded self-assembled nanoparticle-based trimer RBD provides a reference for the design of mRNA vaccines targeting SARS-CoV-2.

Semi- and fully synthetic carbohydrate vaccines against pathogenic bacteria: recent developments.

The importance of vaccine-induced protection was repeatedly demonstrated over the last three decades and emphasized during the recent COVID-19 pandemic as the safest and most effective way of preventing infectious diseases. Vaccines have controlled, and in some cases, eradicated global viral and bacterial infections with high efficiency and at a relatively low cost. Carbohydrates form the capsular sugar coat that surrounds the outer surface of human pathogenic bacteria. Specific surface-exposed bacterial carbohydrates serve as potent vaccine targets that broadened our toolbox against bacterial infections. Since first approved for commercial use, antibacterial carbohydrate-based vaccines mostly rely on inherently complex and heterogenous naturally derived polysaccharides, challenging to obtain in a pure, safe, and cost-effective manner. The introduction of synthetic fragments identical with bacterial capsular polysaccharides provided well-defined and homogenous structures that resolved many challenges of purified polysaccharides. The success of semisynthetic glycoconjugate vaccines against bacterial infections, now in different phases of clinical trials, opened up new possibilities and encouraged further development towards fully synthetic antibacterial vaccine solutions. In this mini-review, we describe the recent achievements in semi- and fully synthetic carbohydrate vaccines against a range of human pathogenic bacteria, focusing on preclinical and clinical studies.

An Epitope Platform for Safe and Effective HTLV-1-Immunization: Potential Applications for mRNA and Peptide-Based Vaccines.

Human T-cell lymphotropic virus type 1 (HTLV-1) infection affects millions of individuals worldwide and can lead to severe leukemia, myelopathy/tropical spastic paraparesis, and numerous other disorders. Pursuing a safe and effective immunotherapeutic approach, we compared the viral polyprotein and the human proteome with a sliding window approach in order to identify oligopeptide sequences unique to the virus. The immunological relevance of the viral unique oligopeptides was assessed by searching them in the immune epitope database (IEDB). We found that HTLV-1 has 15 peptide stretches each consisting of uniquely viral non-human pentapeptides which are ideal candidate for a safe and effective anti-HTLV-1 vaccine. Indeed, experimentally validated HTLV-1 epitopes, as retrieved from the IEDB, contain peptide sequences also present in a vast number of human proteins, thus potentially instituting the basis for cross-reactions. We found a potential for cross-reactivity between the virus and the human proteome and described an epitope platform to be used in order to avoid it, thus obtaining effective, specific, and safe immunization. Potential advantages for mRNA and peptide-based vaccine formulations are discussed.

In Vitro Evaluation of Immunogenicity of Recombinant OMP25 Protein Obtained from Endemic Brucella abortus Biovar 3 as Vaccine Candidate Molecule Against Animal Brucellosis.

BACKGROUND: Brucellosis is a zoonotic disease that causes serious economic losses due to factors, such as miscarriages and decreased milk yield in animals. Existing live vaccines have some disadvantages, so effective vaccines need to be developed with new technological approaches. OBJECTIVE: The primary objectives of this study were the expression and purification of recombinant Omp25 fusion protein from B. abortus, and the evaluation of the effect of the Omp25 protein on cell viability and inflammatory response. METHODS: The omp25 gene region was amplified by a polymerase chain reaction and cloned into a Pet102/D-TOPO expression vector. The protein expression was carried out using the prokaryotic expression system. The recombinant Omp25 protein was purified with affinity chromatography followed by GPC (Gel Permeation Chromatography). The MTS assay and cytokine-release measurements were carried out to evaluate cell viability and inflammatory response, respectively. RESULTS: It was determined that doses of the recombinant Omp25 protein greater than 0.1 µg/mL are toxic to RAW cells. Doses of 1 µg/mL and lower significantly increased inflammation due to Nitric Oxide (NO) levels. ELISA results showed that IFN-γ was produced in stimulated RAW 264.7 cells at a dose that did not affect the viability (0.05 µg/mL). However, IL-12, which is known to have a dual role in the activation of macrophages, did not show a statistically significant difference at the same dose. CONCLUSION: Studies on cell viability and Th1-related cytokine release suggest Omp25 protein to be a promising candidate molecule for vaccine development.

Strategies to load therapeutics into polysaccharide-based nanogels with a focus on microfluidics: A review.

Nowadays nanoparticles are increasingly investigated for the targeted and controlled delivery of therapeutics, as suggested by the high number of research articles (2400 in 2000 vs 8500 in 2020). Among them, almost 2% investigated nanogels in 2020. Nanogels or nanohydrogels (NGs) are nanoparticles formed by a swollen three-dimensional network of synthetic polymers or natural macromolecules such as polysaccharides. NGs represent a highly versatile nanocarrier, able to deliver a number of therapeutics. Currently, NGs are undergoing clinical trials for the delivery of anti-cancer vaccines. Herein, the strategies to load low molecular weight drugs, (poly)peptides and genetic material into polysaccharide NGs as well as to formulate NGs-based vaccines are summarized, with a focus on the microfluidics approach.

An Engineered Receptor-Binding Domain Improves the Immunogenicity of Multivalent SARS-CoV-2 Vaccines.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.

Simultaneous Evaluation of a Vaccine Component Microheterogeneity and Conformational Integrity Using Native Mass Spectrometry and Limited Charge Reduction.

Analytical characterization of extensively modified proteins (such as haptenated carrier proteins in synthetic vaccines) remains a challenging task due to the high degree of structural heterogeneity. Native mass spectrometry (MS) combined with limited charge reduction allows these obstacles to be overcome and enables meaningful characterization of a heavily haptenated carrier protein CRM197 (inactivated diphtheria toxin conjugated with nicotine), a major component of a smoking cessation vaccine. The extensive conjugation results in a near-continuum distribution of ionic signal in electrospray ionization (ESI) mass spectra of haptenated CRM197 even after size-exclusion chromatographic fractionation. However, supplementing the ESI MS measurements with limited charge reduction of ionic populations selected within narrow m/z windows gives rise to well-resolved charge ladders, from which both masses and charge states of the ionic species can be readily deduced. Application of this technique to a research-grade material of CRM197/H7 conjugate not only reveals its marginal conformational stability (manifested by the appearance of high charge-density ions in ESI MS) but also establishes a role of the extent of haptenation as a major factor driving the loss of the higher order structure integrity. The unique information provided by native MS used in combination with limited charge reduction provides a strong argument for this technique to become a standard/required tool in the analytical arsenal in the field of biotechnology and biopharmaceutical analysis, where protein conjugates are becoming increasingly common.

Discovery of Oligosaccharide Antigens for Semi-Synthetic Glycoconjugate Vaccine Leads against Streptococcus suis Serotypes 2, 3, 9 and†14*.

Streptococcus suis bacteria are one of the most serious health problems for pigs and an emerging zoonotic agent in humans working in the swine industry. S. suis bacteria express capsular polysaccharides (CPS) a major bacterial virulence factor that define the serotypes. Oligosaccharides resembling the CPS of S. suis serotypes 2, 3, 9, and 14 have been synthesized, glycans related to serotypes 2 and 9 were placed on glycan array surfaces to screen blood from infected pigs. Lead antigens for the development of semi-synthetic S. suis serotypes 2 and 9 glycoconjugate veterinary vaccines were identified in this way.

Discovery of Semi- and Fully-Synthetic Carbohydrate Vaccines Against Bacterial Infections Using a Medicinal Chemistry Approach.

The glycocalyx, a thick layer of carbohydrates, surrounds the cell wall of most bacterial and parasitic pathogens. Recognition of these unique glycans by the human immune system results in destruction of the invaders. To elicit a protective immune response, polysaccharides either isolated from the bacterial cell surface or conjugated with a carrier protein, for T-cell help, are administered. Conjugate vaccines based on isolated carbohydrates currently protect millions of people against Streptococcus pneumoniae, Haemophilus influenzae type b, and Neisseria meningitides infections. Active pharmaceutical ingredients (APIs) are increasingly discovered by medicinal chemistry and synthetic in origin, rather than isolated from natural sources. Converting vaccines from biologicals to pharmaceuticals requires a fundamental understanding of how the human immune system recognizes carbohydrates and could now be realized. To illustrate the chemistry-based approach to vaccine discovery, I summarize efforts focusing on synthetic glycan-based medicinal chemistry to understand the mammalian antiglycan immune response and define glycan epitopes for novel synthetic glycoconjugate vaccines against Streptococcus pneumoniae, Clostridium difficile, Klebsiella pneumoniae, and other bacteria. The chemical tools described here help us gain fundamental insights into how the human system recognizes carbohydrates and drive the discovery of carbohydrate vaccines.

Design and Synthesis of HCV-E2 Glycoprotein Epitope Mimics in Molecular Construction of Potential Synthetic Vaccines.

Hepatitis C virus remains a global threat, despite the availability of highly effective direct-acting antiviral (DAA) drugs. With thousands of new infections annually, the need for a prophylactic vaccine is evident. However, traditional vaccine design has been unable to provide effective vaccines so far. Therefore, alternative strategies need to be investigated. In this work, a chemistry-based approach is explored towards fully synthetic peptide-based vaccines using epitope mimicry, by focusing on highly effective and conserved amino acid sequences in HCV, which, upon antibody binding, inhibit its bio-activity. Continuous and discontinuous epitope mimics were both chemically synthesized based on the HCV-E2 glycoprotein while using designed fully synthetic cyclic peptides. These cyclic epitope mimics were assembled on an orthogonally protected scaffold. The scaffolded epitope mimics have been assessed in immunization experiments to investigate the elicitation of anti-HCV-E2 glycoprotein antibodies. The neutralizing potential of the elicited antibodies was investigated, representing a first step in employing chemically synthesized epitope mimics as a novel strategy towards vaccine design.

Nanoparticle-Mediated Cytoplasmic Delivery of Messenger RNA Vaccines: Challenges and Future Perspectives.

The COVID-19 pandemic has left scientists and clinicians no choice but a race to find solutions to save lives while controlling the rapid spreading. Messenger RNA (mRNA)-based vaccines have become the front-runners because of their safety profiles, precise and reproducible immune response with more cost-effective and faster production than other types of vaccines. However, the physicochemical properties of naked mRNA necessitate innovative delivery technologies to ferry these 'messengers' to ribosomes inside cells by crossing various barriers and subsequently induce an immune response. Intracellular delivery followed by endosomal escape represents the key strategies for cytoplasmic delivery of mRNA vaccines to the target. This Perspective provides insights into how state-of-the-art nanotechnology helps break the delivery barriers and advance the development of mRNA vaccines. The challenges remaining and future perspectives are outlined.

BNT162b vaccines protect rhesus macaques from SARS-CoV-2.

A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD-foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD-foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD 'down', one-RBD 'up' state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses. Prime-boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2-18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA1-3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728).

Addressing the Cold Reality of mRNA Vaccine Stability.

As mRNA vaccines became the frontrunners in late-stage clinical trials to fight the COVID-19 pandemic, challenges surrounding their formulation and stability became readily apparent. In this commentary, we first describe company proposals, based on available public information, for the (frozen) storage of mRNA vaccine drug products across the vaccine supply chain. We then review the literature on the pharmaceutical stability of mRNA vaccine candidates, including attempts to improve their stability, analytical techniques to monitor their stability, and regulatory guidelines covering product characterization and storage stability. We conclude that systematic approaches to identify the key physicochemical degradation mechanism(s) of formulated mRNA vaccine candidates are currently lacking. Rational design of optimally stabilized mRNA vaccine formulations during storage, transport, and administration at refrigerated or ambient temperatures should thus have top priority in the pharmaceutical development community. In addition to evidence of human immunogenicity against multiple viral pathogens, including compelling efficacy results against COVID-19, another key strength of the mRNA vaccine approach is that it is readily adaptable to rapidly address future outbreaks of new emerging infectious diseases. Consequently, we should not wait for the next pandemic to address and solve the challenges associated with the stability and storage of formulated mRNA vaccines.