CspZ FH-Binding Sites as Epitopes Promote Antibody-Mediated Lyme Borreliae Clearance.

Transmitted by ticks, the bacterium Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD), the most common vector-borne disease in the Northern hemisphere. No effective vaccines are currently available. B. burgdorferi sensu lato produces the CspZ protein that binds to the complement inhibitor, factor H (FH), promoting evasion of the host complement system. We previously showed that while vaccination with CspZ did not protect mice from B. burgdorferi infection, mice can be protected after immunization with CspZ-Y207A/Y211A (CspZ-YA), a CspZ mutant protein without FH-binding activity. To further study the mechanism of this protection, herein we evaluated both poly- and monoclonal antibodies recognizing CspZ FH-binding or non-FH-binding sites. We found that the anti-CspZ antibodies that recognize the FH-binding sites (i.e., block FH-binding activity) eliminate B. burgdorferi sensu lato in vitro more efficiently than those that bind to the non-FH-binding sites, and passive inoculation with anti-FH-binding site antibodies eradicated B. burgdorferi sensu lato in vivo. Antibodies against non-FH-binding sites did not have the same effect. These results emphasize the importance of CspZ FH-binding sites in triggering a protective antibody response against B. burgdorferi sensu lato in future LD vaccines.

Yeast-expressed recombinant SARS-CoV-2 receptor binding domain RBD203-N1 as a COVID-19 protein vaccine candidate.

SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans.

A scalable and reproducible manufacturing process for Phlebotomus papatasi salivary protein PpSP15, a vaccine candidate for leishmaniasis.

Cutaneous leishmaniasis is a parasitic and neglected tropical disease transmitted by the bites of sandflies. The emergence of cutaneous leishmaniasis in areas of war, conflict, political instability, and climate change has prompted efforts to develop a preventive vaccine. One vaccine candidate antigen is PpSP15, a 15 kDa salivary antigen from the sandfly Phlebotomus papatasi that facilitates the infection of the Leishmania parasite and has been shown to induce parasite-specific cell-mediated immunity. Previously, we developed a fermentation process for producing recombinant PpSP15 in Pichia pastoris and a two-chromatographic-step purification process at 100 mL scale. Here we expand the process design to the 10 L scale and examine its reproducibility by performing three identical process runs, an essential transition step towards technology transfer for pilot manufacture. The process was able to reproducibly recover 81% of PpSP15 recombinant protein with a yield of 0.75 g/L of fermentation supernatant, a purity level of 97% and with low variance among runs. Additionally, a freeze-thaw stability study indicated that the PpSP15 recombinant protein remains stable after undergoing three freeze-thaw cycles, and an accelerated stability study confirmed its stability at 37 °C for at least one month. A research cell bank for the expression of PpSP15 was generated and fully characterized. Collectively, the cell bank and the production process are ready for technology transfer for future cGMP pilot manufacturing.

Process development and scale-up optimization of the SARS-CoV-2 receptor binding domain-based vaccine candidate, RBD219-N1C1.

A SARS-CoV-2 RBD219-N1C1 (RBD219-N1C1) recombinant protein antigen formulated on Alhydrogel® has recently been shown to elicit a robust neutralizing antibody response against SARS-CoV-2 pseudovirus in mice. The antigen has been produced under current good manufacturing practices (cGMPs) and is now in clinical testing. Here, we report on process development and scale-up optimization for upstream fermentation and downstream purification of the antigen. This includes production at the 1-L and 5-L scales in the yeast, Pichia pastoris, and the comparison of three different chromatographic purification methods. This culminated in the selection of a process to produce RBD219-N1C1 with a yield of >400 mg per liter of fermentation with >92% purity and >39% target product recovery after purification. In addition, we show the results from analytical studies, including SEC-HPLC, DLS, and an ACE2 receptor binding assay that were performed to characterize the purified proteins to select the best purification process. Finally, we propose an optimized upstream fermentation and downstream purification process that generates quality RBD219-N1C1 protein antigen and is fully scalable at a low cost. KEY POINTS: • Yeast fermentation conditions for a recombinant COVID-19 vaccine were determined. • Three purification protocols for a COVID-19 vaccine antigen were compared. • Reproducibility of a scalable, low-cost process for a COVID-19 vaccine was shown. Graphical abstract.

The Factor H-Binding Site of CspZ as a Protective Target against Multistrain, Tick-Transmitted Lyme Disease.

The spirochete Borrelia burgdorferisensu lato is the causative agent of Lyme disease (LD). The spirochetes produce the CspZ protein that binds to a complement regulator, factor H (FH). Such binding downregulates activation of host complement to facilitate spirochete evasion of complement killing. However, vaccination with CspZ does not protect against LD infection. In this study, we demonstrated that immunization with CspZ-YA, a CspZ mutant protein with no FH-binding activity, protected mice from infection by several spirochete genotypes introduced via tick feeding. We found that the sera from CspZ-YA-vaccinated mice more efficiently eliminated spirochetes and blocked CspZ FH-binding activity than sera from CspZ-immunized mice. We also found that vaccination with CspZ, but not CspZ-YA, triggered the production of anti-FH antibodies, justifying CspZ-YA as an LD vaccine candidate. The mechanistic and efficacy information derived from this study provides insights into the development of a CspZ-based LD vaccine.

A method to probe protein structure from UV absorbance spectra.

Proteins primarily absorb UV light due to the presence of tryptophan, tyrosine, and phenylalanine residues, with absorbance maxima at 280, 275, and 258 nm, respectively. We now demonstrate that a simple value obtained by relating the absorbance at all three wavelengths, [A280/A275 + A280/A258], is a generally useful, robust, and sensitive probe of protein 'foldedness', and thus can be used to investigate unfolding, refolding, disulfide bonds, stability, buffer excipients, and even protein-protein and protein-ligand interactions.

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations.

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.

Biophysical and biochemical characterization of a recombinant Lyme disease vaccine antigen, CspZ-YA.

Lyme disease, caused by Lyme Borrelia spirochetes, is the most common vector-borne illness in the United States. Despite its global significance, with an estimated 14.5 % seroprevalence, there is currently no licensed vaccine. Previously, we demonstrated that CspZ-YA protein conferred protection against Lyme Borrelia infection, making it a promising vaccine candidate. However, such a protein was tagged with hexahistidine, and thus not preferred for vaccine development; furthermore, the formulation to stabilize the protein was understudied. In this work, we developed a two-step purification process for tag-free E. coli-expressed recombinant CspZ-YA. We further utilized various bioassays to analyze the protein and determine the suitable buffer system for long-term storage and formulation as a vaccine immunogen. The results indicated that a buffer with a pH between 6.5 and 8.5 stabilized CspZ-YA by reducing its surface hydrophobicity and colloidal interactions. Additionally, low pH values induced a change in local spatial conformation and resulted in a decrease in α-helix content. Lastly, an optimal salinity of 22-400 mM at pH 7.5 was found to be important for its stability. Collectively, this study provides a fundamental biochemical and biophysical understanding and insights into the ideal stabilizing conditions to produce CspZ-YA recombinant protein for use in vaccine formulation and development.

Functionalized viral nanoparticles as ultrasensitive reporters in lateral-flow assays.

Two types of viral nanoparticles were functionalized with target-specific antibodies and multiple copies of an enzymatic reporter (horseradish peroxidase). The particles were successfully integrated into an immunochromatographic assay detecting MS2 bacteriophage, a model for viral pathogens. The sensitivity of the assay was greatly superior to conventional gold nanoparticle lateral flow assays, and results could easily be evaluated, even without advanced lab instruments.

From concept to delivery: a yeast-expressed recombinant protein-based COVID-19 vaccine technology suitable for global access.

INTRODUCTION: The development of a yeast-expressed recombinant protein-based vaccine technology co-developed with LMIC vaccine producers and suitable as a COVID-19 vaccine for global access is described. The proof-of-concept for developing a SARS-CoV-2 spike protein receptor-binding domain (RBD) antigen as a yeast-derived recombinant protein vaccine technology is described. AREAS COVERED: Genetic Engineering: The strategy is presented for the design and genetic modification used during cloning and expression in the yeast system. Process and Assay Development: A summary is presented of how a scalable, reproducible, and robust production process for the recombinant protein COVID-19 vaccine antigen was developed. Formulation and Pre-clinical Strategy: We report on the pre-clinical and formulation strategy used for the proof-of-concept evaluation of the SARS-CoV-2 RBD vaccine antigen. Technology Transfer and Partnerships: The process used for the technology transfer and co-development with LMIC vaccine producers is described. Clinical Development and Delivery: The approach used by LMIC developers to establish the industrial process, clinical development, and deployment is described. EXPERT OPINION: Highlighted is an alternative model for developing new vaccines for emerging infectious diseases of pandemic importance starting with an academic institution directly transferring their technology to LMIC vaccine producers without the involvement of multinational pharma companies.

A Recombinant Protein XBB.1.5 RBD/Alum/CpG Vaccine Elicits High Neutralizing Antibody Titers against Omicron Subvariants of SARS-CoV-2.

(1) Background: We previously reported the development of a recombinant protein SARS-CoV-2 vaccine, consisting of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, adjuvanted with aluminum hydroxide (alum) and CpG oligonucleotides. In mice and non-human primates, our wild-type (WT) RBD vaccine induced high neutralizing antibody titers against the WT isolate of the virus, and, with partners in India and Indonesia, it was later developed into two closely resembling human vaccines, Corbevax and Indovac. Here, we describe the development and characterization of a next-generation vaccine adapted to the recently emerging XBB variants of SARS-CoV-2. (2) Methods: We conducted preclinical studies in mice using a novel yeast-produced SARS-CoV-2 XBB.1.5 RBD subunit vaccine candidate formulated with alum and CpG. We examined the neutralization profile of sera obtained from mice vaccinated twice intramuscularly at a 21-day interval with the XBB.1.5-based RBD vaccine, against WT, Beta, Delta, BA.4, BQ.1.1, BA.2.75.2, XBB.1.16, XBB.1.5, and EG.5.1 SARS-CoV-2 pseudoviruses. (3) Results: The XBB.1.5 RBD/CpG/alum vaccine elicited a robust antibody response in mice. Furthermore, the serum from vaccinated mice demonstrated potent neutralization against the XBB.1.5 pseudovirus as well as several other Omicron pseudoviruses. However, regardless of the high antibody cross-reactivity with ELISA, the anti-XBB.1.5 RBD antigen serum showed low neutralizing titers against the WT and Delta virus variants. (4) Conclusions: Whereas we observed modest cross-neutralization against Omicron subvariants with the sera from mice vaccinated with the WT RBD/CpG/Alum vaccine or with the BA.4/5-based vaccine, the sera raised against the XBB.1.5 RBD showed robust cross-neutralization. These findings underscore the imminent opportunity for an updated vaccine formulation utilizing the XBB.1.5 RBD antigen.

Fluorescence correlation spectroscopy study of protein transport and dynamic interactions with clustered-charge peptide adsorbents.

Ion-exchange chromatography relies on electrostatic interactions between the adsorbent and the adsorbate and is used extensively in protein purification. Conventional ion-exchange chromatography uses ligands that are singly charged and randomly dispersed over the adsorbent, creating a heterogeneous distribution of potential adsorption sites. Clustered-charge ion exchangers exhibit higher affinity, capacity, and selectivity than their dispersed-charge counterparts of the same total charge density. In the present work, we monitored the transport behavior of an anionic protein near clustered-charge adsorbent surfaces using fluorescence correlation spectroscopy. We can resolve protein-free diffusion, hindered diffusion, and association with bare glass, agarose-coated, and agarose-clustered peptide surfaces, demonstrating that this method can be used to understand and ultimately optimize clustered-charge adsorbent and other surface interactions at the molecular scale.

Past, present, and future of Lyme disease vaccines: antigen engineering approaches and mechanistic insights.

INTRODUCTION: Transmitted by ticks, Lyme disease is the most common vector-borne disease in the Northern hemisphere. Despite the geographical expansion of human Lyme disease cases, no effective preventive strategies are currently available. Developing an efficacious and safe vaccine is therefore urgently needed. Efforts have previously been taken to identify vaccine targets in the causative pathogen (Borrelia burgdorferi sensu lato) and arthropod vector (Ixodes spp.). However, progress was impeded due to a lack of consumer confidence caused by the myth of undesired off-target responses, low immune responses, a limited breadth of immune reactivity, as well as by the complexities of the vaccine process development. AREA COVERED: In this review, we summarize the antigen engineering approaches that have been applied to overcome those challenges and the underlying mechanisms that can be exploited to improve both safety and efficacy of future Lyme disease vaccines. EXPERT OPINION: Over the past two decades, several new genetically redesigned Lyme disease vaccine candidates have shown success in both preclinical and clinical settings and built a solid foundation for further development. These studies have greatly informed the protective mechanisms of reducing Lyme disease burdens and ending the endemic of this disease.

A novel multi-epitope recombinant protein elicits an antigen-specific CD8+ T cells response in Trypanosoma cruzi-infected mice.

About 6.5 million people worldwide are afflicted by Chagas disease, which is caused by the protozoan parasite Trypanosoma cruzi. The development of a therapeutic vaccine to prevent the progression of Chagasic cardiomyopathy has been proposed as an alternative for antiparasitic chemotherapy. Bioinformatics tools can predict MHC class I CD8 + epitopes for inclusion in a single recombinant protein with the goal to develop a multivalent vaccine. We expressed a novel recombinant protein Tc24-C4.10E harboring ten nonameric CD8 + epitopes and using Tc24-C4 protein as scaffold to evaluate the therapeutic effect in acute T. cruzi infection. T. cruzi-infected mice were immunized with Tc24-C4.10E or Tc24-C4 in a 50-day model of acute infection. Tc24-C4.10E-treated mice showed a decreased parasitemia compared to the Tc24-C4 (non-adjuvant) immunized mice or control group. Moreover, Tc24-C4.10E induced a higher stimulation index of CD8 + T cells producing IFNγ and IL-4 cytokines. These results suggest that the addition of the MHC Class I epitopes to Tc24-C4 can synergize the antigen-specific cellular immune responses, providing proof-of-concept that this approach could lead to the development of a promising vaccine candidate for Chagas disease.

Receptor-binding domain recombinant protein on alum-CpG induces broad protection against SARS-CoV-2 variants of concern.

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the CorbevaxTM vaccine that recently received emergency use authorization by the Drugs Controller General ofIndia. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum + CpG. We also evaluated mice immunized with RBD/alum + CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum + CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum + CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2, including variants of concern.

Receptor-binding domain recombinant protein on alum-CpG induces broad protection against SARS-CoV-2 variants of concern.

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the CorbevaxTM vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum+CpG. We also evaluated mice immunized with RBD/alum+CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum+CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum+CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2 including variants of concern.

Recombinant protein vaccines, a proven approach against coronavirus pandemics.

With the COVID-19 pandemic now ongoing for close to a year, people all over the world are still waiting for a vaccine to become available. The initial focus of accelerated global research and development efforts to bring a vaccine to market as soon as possible was on novel platform technologies that promised speed but had limited history in the clinic. In contrast, recombinant protein vaccines, with numerous examples in the clinic for many years, missed out on the early wave of investments from government and industry. Emerging data are now surfacing suggesting that recombinant protein vaccines indeed might offer an advantage or complement to the nucleic acid or viral vector vaccines that will likely reach the clinic faster. Here, we summarize the current public information on the nature and on the development status of recombinant subunit antigens and adjuvants targeting SARS-CoV-2 infections.

SARS-CoV-2 RBD219-N1C1: A Yeast-Expressed SARS-CoV-2 Recombinant Receptor-Binding Domain Candidate Vaccine Stimulates Virus Neutralizing Antibodies and T-cell Immunity in Mice.

There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast ( Pichia pastoris ), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel ® were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ, IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel ® containing formulation and possibly in combination with other immunostimulants.

SARS­CoV-2 RBD219-N1C1: A yeast-expressed SARS-CoV-2 recombinant receptor-binding domain candidate vaccine stimulates virus neutralizing antibodies and T-cell immunity in mice.

There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here, we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel® were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel® containing formulation and possibly in combination with other immunostimulants.

Genetic modification to design a stable yeast-expressed recombinant SARS-CoV-2 receptor binding domain as a COVID-19 vaccine candidate.

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed. METHOD: We constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a "wild type" RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. RESULTS AND CONCLUSIONS: These three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation. GENERAL SIGNIFICANCE: By genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.