Elucidation of novel compounds and epitope-based peptide vaccine design against C30 endopeptidase regions of SARS-CoV-2 using immunoinformatics approaches.

There has been progressive improvement in immunoinformatics approaches for epitope-based peptide design. Computational-based immune-informatics approaches were applied to identify the epitopes of SARS-CoV-2 to develop vaccines. The accessibility of the SARS-CoV-2 protein surface was analyzed, and hexa-peptide sequences (KTPKYK) were observed having a maximum score of 8.254, located between amino acids 97 and 102, whereas the FSVLAC at amino acids 112 to 117 showed the lowest score of 0.114. The surface flexibility of the target protein ranged from 0.864 to 1.099 having amino acid ranges of 159 to 165 and 118 to 124, respectively, harboring the FCYMHHM and YNGSPSG hepta-peptide sequences. The surface flexibility was predicted, and a 0.864 score was observed from amino acids 159 to 165 with the hepta-peptide (FCYMHHM) sequence. Moreover, the highest score of 1.099 was observed between amino acids 118 and 124 against YNGSPSG. B-cell epitopes and cytotoxic T-lymphocyte (CTL) epitopes were also identified against SARS-CoV-2. In molecular docking analyses, -0.54 to -26.21 kcal/mol global energy was observed against the selected CTL epitopes, exhibiting binding solid energies of -3.33 to -26.36 kcal/mol. Based on optimization, eight epitopes (SEDMLNPNY, GSVGFNIDY, LLEDEFTPF, DYDCVSFCY, GTDLEGNFY, QTFSVLACY, TVNVLAWLY, and TANPKTPKY) showed reliable findings. The study calculated the associated HLA alleles with MHC-I and MHC-II and found that MHC-I epitopes had higher population coverage (0.9019% and 0.5639%) than MHC-II epitopes, which ranged from 58.49% to 34.71% in Italy and China, respectively. The CTL epitopes were docked with antigenic sites and analyzed with MHC-I HLA protein. In addition, virtual screening was conducted using the ZINC database library, which contained 3,447 compounds. The 10 top-ranked scrutinized molecules (ZINC222731806, ZINC077293241, ZINC014880001, ZINC003830427, ZINC030731133, ZINC003932831, ZINC003816514, ZINC004245650, ZINC000057255, and ZINC011592639) exhibited the least binding energy (-8.8 to -7.5 kcal/mol). The molecular dynamics (MD) and immune simulation data suggest that these epitopes could be used to design an effective SARS-CoV-2 vaccine in the form of a peptide-based vaccine. Our identified CTL epitopes have the potential to inhibit SARS-CoV-2 replication.

Immunoinformatics Approaches in Designing Vaccines Against COVID-19.

Since the onset of the COVID-19 pandemic, a number of approaches have been adopted by the scientific communities for developing efficient vaccine candidate against SARS-CoV-2. Conventional approaches of developing a vaccine require a long time and a series of trials and errors which indeed limit the feasibility of such approaches for developing a dependable vaccine in an emergency situation like the COVID-19 pandemic. Hitherto, most of the available vaccines have been developed against a particular antigen of SARS-CoV, spike protein in most of the cases, and intriguingly, these vaccines are not effective against all the pathogenic coronaviruses. In this context, immunoinformatics-based reverse vaccinology approaches enable a robust design of efficacious peptide-based vaccines against all the infectious strains of coronaviruses within a short frame of time. In this chapter, we enumerate the methodological trajectory of developing a universal anti-SARS-CoV-2 vaccine, namely, "AbhiSCoVac," through advanced computational biology-based immunoinformatics approach and its in-silico validation using molecular dynamics simulations.

A novel mutation-proof, next-generation vaccine to fight against upcoming SARS-CoV-2 variants and subvariants, designed through AI enabled approaches and tools, along with the machine learning based immune simulation: A vaccine breakthrough.

Emerging SARS-CoV-2 variants and subvariants are great concerns for their significant mutations, which are also responsible for vaccine escape. Therefore, the study was undertaken to develop a mutation-proof, next-generation vaccine to protect against all upcoming SARS-CoV-2 variants. We used advanced computational and bioinformatics approaches to develop a multi-epitopic vaccine, especially the AI model for mutation selection and machine learning (ML) strategies for immune simulation. AI enabled and the top-ranked antigenic selection approaches were used to select nine mutations from 835 RBD mutations. We selected twelve common antigenic B cell and T cell epitopes (CTL and HTL) containing the nine RBD mutations and joined them with the adjuvants, PADRE sequence, and suitable linkers. The constructs' binding affinity was confirmed through docking with TLR4/MD2 complex and showed significant binding free energy (-96.67 kcal mol-1) with positive binding affinity. Similarly, the calculated eigenvalue (2.428517e-05) from the NMA of the complex reveals proper molecular motion and superior residues' flexibility. Immune simulation shows that the candidate can induce a robust immune response. The designed mutation-proof, multi-epitopic vaccine could be a remarkable candidate for upcoming SARS-CoV-2 variants and subvariants. The study method might guide researchers in developing AI-ML and immunoinformatics-based vaccines for infectious disease.

Multi-pathogen based chimeric vaccine to fight against COVID-19 and concomitant coinfections.

BACKGROUND: COVID-19 has proved to be a fatal disease of the year 2020, due to which thousands of people globally have lost their lives, and still, the infection cases are at a high rate. Experimental studies suggested that SARS-CoV-2 interacts with various microorganisms, and this coinfection is accountable for the augmentation of infection severity. METHODS AND RESULTS: In this study, we have designed a multi-pathogen vaccine by involving the immunogenic proteins from S. pneumonia, H. influenza, and M. tuberculosis, as they are dominantly associated with SARS-CoV-2. A total of 8 antigenic protein sequences were selected to predict B-cell, HTL, and CTL epitopes restricted to the most prevalent HLA alleles. The selected epitopes were antigenic, non-allergenic, and non-toxic and were linked with adjuvant and linkers to make the vaccine protein more immunogenic, stable, and flexible. The tertiary structure, Ramachandran plot, and discontinuous B-cell epitopes were predicted. Docking and MD simulation study has shown efficient binding of the chimeric vaccine with the TLR4 receptor. CONCLUSION: The in silico immune simulation analysis has shown a high level of cytokines and IgG after a three-dose injection. Hence, this strategy could be a better way to decrease the disease's severity and could be used as a weapon to prevent this pandemic.

In silico EsxG EsxH rational epitope selection: Candidate epitopes for vaccine design against pulmonary tuberculosis.

Rational design of new vaccines against pulmonary tuberculosis is imperative. Early secreted antigens (Esx) G and H are involved in metal uptake, drug resistance, and immune response evasion. These characteristics make it an ideal target for rational vaccine development. The aim of this study is to show the rational design of epitope-based peptide vaccines by using bioinformatics and structural vaccinology tools. A total of 4.15 µs of Molecular Dynamics simulations were carried out to describe the behavior in solution of heterodimer, single epitopes, and epitopes loaded into MHC-II complexes. In order to predict T and B cell epitopes for antigenic activation, bioinformatic tools were used. Hence, we propose three epitopes with the potential to design pulmonary tuberculosis vaccines. The possible use of the proposed epitopes includes subunit vaccines, as a booster in BCG vaccination to improve its immune response, as well as the generation of antibodies that interfere with the Mycobacterium tuberculosis homeostasis, affecting its survival.

Vaccine Adjuvants: Selection Criteria, Mechanism of Action Associated with Immune Responses and Future Directions.

The most effective method to minimize the prevalence of infectious diseases is vaccination. Vaccines enhance immunity and provide protection against different kinds of infections. Subunit vaccines are safe and less toxic, but due to their lower immunogenicity, they need adjuvants to boost the immune system. Adjuvants are small particles/molecules integrated into a vaccine to enhance the immunogenic feedback of antigens. They play a significant role to enhance the potency and efficiency of vaccines. There are several types of adjuvants with different mechanisms of action; therefore, improved knowledge of their immunogenicity will help develop a new generation of adjuvants. Many trials have been designed using different kinds of vaccine adjuvants to examine their safety and efficacy, but in practice, only a few have entered in animal and human clinical trials. However, for the development of safe and effective vaccines, it is important to have adequate knowledge of the side effects and toxicity of different adjuvants. The current review discussed the adjuvants which are available for producing modern vaccines as well as some new classes of adjuvants in clinical trials.

Subunit vaccines with a saponin-based adjuvant boost humoral and cellular immunity to MERS coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) outbreaks have constituted a public health issue with drastic mortality higher than 34%, necessitating the development of an effective vaccine. During MERS-CoV infection, the trimeric spike protein on the viral envelope is primarily responsible for attachment to host cellular receptor, dipeptidyl peptidase 4 (DPP4). With the goal of generating a protein-based prophylactic, we designed a subunit vaccine comprising the recombinant S1 protein with a trimerization motif (S1-Fd) and examined its immunogenicity and protective immune responses in combination with various adjuvants. We found that sera from immunized wild-type and human DPP4 transgenic mice contained S1-specific antibodies that can neutralize MERS-CoV infection in susceptible cells. Vaccination with S1-Fd protein in combination with a saponin-based QS-21 adjuvant provided long-term humoral as well as cellular immunity in mice. Our findings highlight the significance of the trimeric S1 protein in the development of MERS-CoV vaccines and offer a suitable adjuvant, QS-21, to induce robust and prolonged memory T cell response.

Nanostrategies to Develop Current Antiviral Vaccines.

Infectious diseases are a worldwide concern. They are responsible for increasing the mortality rate and causing economic and social problems. Viral epidemics and pandemics, such as the COVID-19 pandemic, force the scientific community to consider molecules with antiviral activity. A number of viral infections still do not have a vaccine or efficient treatment and it is imperative to search for vaccines to control these infections. In this context, nanotechnology in association with the design of vaccines has presented an option for virus control. Nanovaccines have displayed an impressive immune response using a low dosage. This review aims to describe the advances and update the data in studies using nanovaccines and their immunomodulatory effect against human viruses.

A CHO stable pool production platform for rapid clinical development of trimeric SARS-CoV-2 spike subunit vaccine antigens.

Protein expression from stably transfected Chinese hamster ovary (CHO) clones is an established but time-consuming method for manufacturing therapeutic recombinant proteins. The use of faster, alternative approaches, such as non-clonal stable pools, has been restricted due to lower productivity and longstanding regulatory guidelines. Recently, the performance of stable pools has improved dramatically, making them a viable option for quickly producing drug substance for GLP-toxicology and early-phase clinical trials in scenarios such as pandemics that demand rapid production timelines. Compared to stable CHO clones which can take several months to generate and characterize, stable pool development can be completed in only a few weeks. Here, we compared the productivity and product quality of trimeric SARS-CoV-2 spike protein ectodomains produced from stable CHO pools or clones. Using a set of biophysical and biochemical assays we show that product quality is very similar and that CHO pools demonstrate sufficient productivity to generate vaccine candidates for early clinical trials. Based on these data, we propose that regulatory guidelines should be updated to permit production of early clinical trial material from CHO pools to enable more rapid and cost-effective clinical evaluation of potentially life-saving vaccines.

Application of Traditional Vaccine Development Strategies to SARS-CoV-2.

Over the past 150 years, vaccines have revolutionized the relationship between people and disease. During the COVID-19 pandemic, technologies such as mRNA vaccines have received attention due to their novelty and successes. However, more traditional vaccine development platforms have also yielded important tools in the worldwide fight against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A variety of approaches have been used to develop COVID-19 vaccines that are now authorized for use in countries around the world. In this review, we highlight strategies that focus on the viral capsid and outwards, rather than on the nucleic acids inside. These approaches fall into two broad categories: whole-virus vaccines and subunit vaccines. Whole-virus vaccines use the virus itself, in either an inactivated or an attenuated state. Subunit vaccines contain instead an isolated, immunogenic component of the virus. Here, we highlight vaccine candidates that apply these approaches against SARS-CoV-2 in different ways. In a companion article (H. M. Rando, R. Lordan, L. Kolla, E. Sell, et al., mSystems 8:e00928-22, 2023, https://doi.org/10.1128/mSystems.00928-22), we review the more recent and novel development of nucleic acid-based vaccine technologies. We further consider the role that these COVID-19 vaccine development programs have played in prophylaxis at the global scale. Well-established vaccine technologies have proved especially important to making vaccines accessible in low- and middle-income countries. Vaccine development programs that use established platforms have been undertaken in a much wider range of countries than those using nucleic acid-based technologies, which have been led by wealthy Western countries. Therefore, these vaccine platforms, though less novel from a biotechnological standpoint, have proven to be extremely important to the management of SARS-CoV-2. IMPORTANCE The development, production, and distribution of vaccines is imperative to saving lives, preventing illness, and reducing the economic and social burdens caused by the COVID-19 pandemic. Vaccines that use cutting-edge biotechnology have played an important role in mitigating the effects of SARS-CoV-2. However, more traditional methods of vaccine development that were refined throughout the 20th century have been especially critical to increasing vaccine access worldwide. Effective deployment is necessary to reducing the susceptibility of the world's population, which is especially important in light of emerging variants. In this review, we discuss the safety, immunogenicity, and distribution of vaccines developed using established technologies. In a separate review, we describe the vaccines developed using nucleic acid-based vaccine platforms. From the current literature, it is clear that the well-established vaccine technologies are also highly effective against SARS-CoV-2 and are being used to address the challenges of COVID-19 globally, including in low- and middle-income countries. This worldwide approach is critical for reducing the devastating impact of SARS-CoV-2.

Evaluation of the Mucosal Immunity Effect of Bovine Viral Diarrhea Virus Subunit Vaccine E2Fc and E2Ft.

Classified as a class B infectious disease by the World Organization for Animal Health (OIE), bovine viral diarrhea/mucosal disease is an acute, highly contagious disease caused by the bovine viral diarrhea virus (BVDV). Sporadic endemics of BVDV often lead to huge economic losses to the dairy and beef industries. To shed light on the prevention and control of BVDV, we developed two novel subunit vaccines by expressing bovine viral diarrhea virus E2 fusion recombinant proteins (E2Fc and E2Ft) through suspended HEK293 cells. We also evaluated the immune effects of the vaccines. The results showed that both subunit vaccines induced an intense mucosal immune response in calves. Mechanistically, E2Fc bonded to the Fc γ receptor (FcγRI) on antigen-presenting cells (APCs) and promoted IgA secretion, leading to a stronger T-cell immune response (Th1 type). The neutralizing antibody titer stimulated by the mucosal-immunized E2Fc subunit vaccine reached 1:64, which was higher than that of the E2Ft subunit vaccine and that of the intramuscular inactivated vaccine. The two novel subunit vaccines for mucosal immunity developed in this study, E2Fc and E2Ft, can be further used as new strategies to control BVDV by enhancing cellular and humoral immunity.

Protection from COVID-19 disease in hamsters vaccinated with subunit SARS-CoV-2 S1 mucosal vaccines adjuvanted with different adjuvants.

Introduction: Adjuvant plays an important role in directing the immune responses induced by vaccines. In previous studies, we have shown that a mucosal SARS-CoV-2 S1 subunit vaccine adjuvanted with a combination of CpG, Poly I:C and IL-15 (named CP15) induced effective mucosal and systemic immunity and conferred nearly sterile protection against SARS-CoV-2 viral replication in macaque models. Methods: In this study, we used a hamster model, which mimics the human scenario and reliably exhibits severe SARS-CoV-2 disease similar to hospitalized patients, to investigate the protection efficacy of the vaccines against COVID-19 disease. We compared the weight loss, viral loads (VLs), and clinical observation scores of three different vaccine regimens. All three regimens consisted of priming/boosting with S1 subunit vaccines, but adjuvanted with alum and/or CP15 administrated by either intramuscular (IM) or intranasal (IN) routes: Group 1 was adjuvanted with alum/alum administrated IM/IM; Group 2 was alum-IM/CP15-IN; and Group 3 was CP15-IM/CP15-IN. Results: After challenge with SARS-CoV-2 WA strain, we found that the alum/CP15 group showed best protection against weight loss, while the CP15 group demonstrated best reduction of oral SARS-CoV-2 VLs, suggesting that the protection profiles were different. Sex differences for VL and clinical scores were observed. Humoral immunity was induced but not correlated with protection. Moreover, S1-specific binding antibody titers against beta, omicron BA.1, and BA.2 variants showed 2.6-, 4.9- and 2.8- fold reduction, respectively, compared to the Wuhan strain. Discussion: Overall, the data suggested that adjuvants in subunit vaccines determine the protection profiles after SARS-CoV-2 infection and that nasal/oral mucosal immunization can protect against systemic COVID-19 disease.

Precise Epitope Organization with Self-adjuvant Framework Nucleic Acid for Efficient COVID-19 Peptide Vaccine Construction.

Peptide vaccines have advantages in easy fabrication and high safety, but their effectiveness is hampered by the poor immunogenicity of the epitopes themselves. Herein, we constructed a series of framework nucleic acids (FNAs) with regulated rigidity and size to precisely organize epitopes in order to reveal the influence of epitope spacing and carrier rigidity on the efficiency of peptide vaccines. We found that assembling epitopes on rigid tetrahedral FNAs (tFNAs) with the appropriate size could efficiently enhance their immunogenicity. Further, by integrating epitopes from SARS-CoV-2 on preferred tFNAs, we constructed a COVID-19 peptide vaccine which could induce high titers of IgG against the receptor binding domain (RBD) of SARS-CoV-2 spike protein and increase the ratio of memory B and T cells in mice. Considering the good biocompatibility of tFNAs, our research provides a new idea for developing efficient peptide vaccines against viruses and possibly other diseases.

Immunoinformatics-aided design of a new multi-epitope vaccine adjuvanted with domain 4 of pneumolysin against Streptococcus pneumoniae strains.

BACKGROUND: Streptococcus pneumoniae (Pneumococcus) has remained a leading cause of fatal infections such as pneumonia, meningitis, and sepsis. Moreover, this pathogen plays a major role in bacterial co-infection in patients with life-threatening respiratory virus diseases such as influenza and COVID-19. High morbidity and mortality in over one million cases, especially in very young children and the elderly, are the main motivations for pneumococcal vaccine development. Due to the limitations of the currently marketed polysaccharide-based vaccines, non-serotype-specific protein-based vaccines have received wide research interest in recent years. One step further is to identify high antigenic regions within multiple highly-conserved proteins in order to develop peptide vaccines that can affect various stages of pneumococcal infection, providing broader serotype coverage and more effective protection. In this study, immunoinformatics tools were used to design an effective multi-epitope vaccine in order to elicit neutralizing antibodies against multiple strains of pneumococcus. RESULTS: The B- and T-cell epitopes from highly protective antigens PspA (clades 1-5) and PhtD were predicted and immunodominant peptides were linked to each other with proper linkers. The domain 4 of Ply, as a potential TLR4 agonist adjuvant candidate, was attached to the end of the construct to enhance the immunogenicity of the epitope vaccine. The evaluation of the physicochemical and immunological properties showed that the final construct was stable, soluble, antigenic, and non-allergenic. Furthermore, the protein was found to be acidic and hydrophilic in nature. The protein 3D-structure was built and refined, and the Ramachandran plot, ProSA-web, ERRAT, and Verify3D validated the quality of the final model. Molecular docking analysis showed that the designed construct via Ply domain 4 had a strong interaction with TLR4. The structural stability of the docked complex was confirmed by molecular dynamics. Finally, codon optimization was performed for gene expression in E. coli, followed by in silico cloning in the pET28a(+) vector. CONCLUSION: The computational analysis of the construct showed acceptable results, however, the suggested vaccine needs to be experimentally verified in laboratory to ensure its safety and immunogenicity.

[Preparation and immungenicity of recombinant protein containing intramolecular adjuvant in SARS-CoV-2 RBD domain].

A fusion protein containing a tetanus toxin peptide, a tuftsin peptide and a SARS-CoV-2S protein receptor-binding domain (RBD) was prepared to investigate the effect of intramolecular adjuvant on humoral and cellular immunity of RBD protein. The tetanus toxin peptide, tuftsin peptide and S protein RBD region were connected by a flexible polypeptide, and a recombinant vector was constructed after codon optimization. The recombinant S-TT-tuftsin protein was prepared by prokaryotic expression and purification. BALB/c mice were immunized after mixed with aluminum adjuvant, and the humoral and cellular immune effects were evaluated. The recombinant S-TT-tuftsin protein was expressed as an inclusion body, and was purified by ion exchange chromatography and renaturated by gradient dialysis. The renaturated protein was identified by Dot blotting and reacted with serum of descendants immunized with SARS-CoV-2 subunit vaccine. The results showed that the antibody level reached a plateau after 35 days of immunization, and the serum antibody ELISA titer of mice immunized with recombinant protein containing intramolecular adjuvant was up to 1:66 240, which was significantly higher than that of mice immunized with S-RBD protein (P < 0.05). At the same time, the recombinant protein containing intramolecular adjuvant stimulated mice to produce a stronger lymphocyte proliferation ability. The stimulation index was 4.71±0.15, which was significantly different from that of the S-RBD protein (1.83±0.09) (P < 0.000 1). Intramolecular adjuvant tetanus toxin peptide and tuftsin peptide significantly enhanced the humoral and cellular immune effect of the SARS-CoV-2 S protein RBD domain, which provideda theoretical basis for the development of subunit vaccines for SARS-CoV-2 and other viruses.

T cell immunity ameliorates COVID-19 disease severity and provides post-exposure prophylaxis after peptide-vaccination, in Syrian hamsters.

Background: The emergence of novel SARS-CoV-2 variants that resist neutralizing antibodies drew the attention to cellular immunity and calls for the development of alternative vaccination strategies to combat the pandemic. Here, we have assessed the kinetics of T cell responses and protective efficacy against severe COVID-19 in pre- and post-exposure settings, elicited by PolyPEPI-SCoV-2, a peptide based T cell vaccine. Methods: 75 Syrian hamsters were immunized subcutaneously with PolyPEPI-SCoV-2 on D0 and D14. On D42, hamsters were intranasally challenged with 102 TCID50 of the virus. To analyze immunogenicity by IFN-γ ELISPOT and antibody secretion, lymphoid tissues were collected both before (D0, D14, D28, D42) and after challenge (D44, D46, D49). To measure vaccine efficacy, lung tissue, throat swabs and nasal turbinate samples were assessed for viral load and histopathological changes. Further, body weight was monitored on D0, D28, D42 and every day after challenge. Results: The vaccine induced robust activation of T cells against all SARS-CoV-2 structural proteins that were rapidly boosted after virus challenge compared to control animals (~4-fold, p<0.05). A single dose of PolyPEPI-SCoV-2 administered one day after challenge also resulted in elevated T cell response (p<0.01). The vaccination did not induce virus-specific antibodies and viral load reduction. Still, peptide vaccination significantly reduced body weight loss (p<0.001), relative lung weight (p<0.05) and lung lesions (p<0.05), in both settings. Conclusion: Our study provides first proof of concept data on the contribution of T cell immunity on disease course and provide rationale for the use of T cell-based peptide vaccines against both novel SARS-CoV-2 variants and supports post-exposure prophylaxis as alternative vaccination strategy against COVID-19.

A Structural View at Vaccine Development against M. tuberculosis.

Tuberculosis (TB) is still the leading global cause of death from an infectious bacterial agent. Limiting tuberculosis epidemic spread is therefore an urgent global public health priority. As stated by the WHO, to stop the spread of the disease we need a new vaccine, with better coverage than the current Mycobacterium bovis BCG vaccine. This vaccine was first used in 1921 and, since then, there are still no new licensed tuberculosis vaccines. However, there is extremely active research in the field, with a steep acceleration in the past decades, due to the advance of technologies and more rational vaccine design strategies. This review aims to gather latest updates in vaccine development in the various clinical phases and to underline the contribution of Structural Vaccinology (SV) to the development of safer and effective antigens. In particular, SV and the development of vaccine adjuvants is making the use of subunit vaccines, which are the safest albeit the less antigenic ones, an achievable goal. Indeed, subunit vaccines overcome safety concerns but need to be rationally re-engineered to enhance their immunostimulating effects. The larger availability of antigen structural information as well as a better understanding of the complex host immune response to TB infection is a strong premise for a further acceleration of TB vaccine development.

Meeting vaccine formulation challenges in an emergency setting: Towards the development of accessible vaccines.

Vaccination is considered one of the most successful strategies to prevent infectious diseases. In the event of a pandemic or epidemic, the rapid development and distribution of the vaccine to the population is essential to reduce mortality, morbidity and transmission. As seen during the COVID-19 pandemic, the production and distribution of vaccines has been challenging, in particular for resource-constrained settings, essentially slowing down the process of achieving global coverage. Pricing, storage, transportation and delivery requirements of several vaccines developed in high-income countries resulted in limited access for low-and-middle income countries (LMICs). The capacity to manufacture vaccines locally would greatly improve global vaccine access. In particular, for the development of classical subunit vaccines, the access to vaccine adjuvants is a pre-requisite for more equitable access to vaccines. Vaccine adjuvants are agents required to augment or potentiate, and possibly target the specific immune response to such type of vaccine antigens. Openly accessible or locally produced vaccine adjuvants may allow for faster immunization of the global population. For local research and development of adjuvanted vaccines to expand, knowledge on vaccine formulation is of paramount importance. In this review, we aim to discuss the optimal characteristics of a vaccine developed in an emergency setting by focusing on the importance of vaccine formulation, appropriate use of adjuvants and how this may help overcome barriers for vaccine development and production in LMICs, achieve improved vaccine regimens, delivery and storage requirements.

Development of an ELISA Assay for the Determination of SARS-CoV-2 Protein Subunit Vaccine Antigen Content.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein subunit vaccine is one of the mainstream technology platforms for the development of COVID-19 vaccines, and most R&D units use the receptor-binding domain (RBD) or spike (S) protein as the main target antigen. The complexity of vaccine design, sequence, and expression systems makes it urgent to establish common antigen assays to facilitate vaccine development. In this study, we report the development of a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the antigen content of SARS-CoV-2 protein subunit vaccines based on the United States Pharmacopeia <1220> and ICH (international conference on harmonization) Q14 and Q2 (R2) requirements. A monoclonal antibody (mAb), 20D8, was identified as the detection antibody based on its high RBD binding activity (EC50 = 8.4 ng/mL), broad-spectrum anti-variant neutralizing activity (EC50: 2.7−9.8 ng/mL for pseudovirus and EC50: 9.6−127 ng/mL for authentic virus), good in vivo protection, and a recognized linear RBD epitope (369−379 aa). A porcine anti-RBD polyclonal antibody was selected as the coating antibody. Assay performance met the requirements of the analytical target profile with an accuracy and precision of ≥90% and adequate specificity. Within the specification range of 70−143%, the method capability index was >0.96; the misjudgment probability was <0.39%. The method successfully detected SARS-CoV-2 protein subunit vaccine antigens (RBD or S protein sequences in Alpha, Beta, Gamma, or Delta variants) obtained from five different manufacturers. Thus, we present a new robust, reliable, and general method for measuring the antigenic content of SARS-CoV-2 protein subunit vaccines. In addition to currently marketed and emergency vaccines, it is suitable for vaccines in development containing antigens derived from pre-Omicron mutant strains.