Two formulations of coronavirus disease-19 recombinant subunit vaccine candidate made up of S1 fragment protein P1, S2 fragment protein P2, and nucleocapsid protein elicit strong immunogenicity in mice.

INTRODUCTION: Coronavirus disease (COVID-19) is ongoing as a global epidemic and there is still a need to develop much safer and more effective new vaccines that can also be easily adapted to important variants of the pathogen. In the present study in this direction, we developed a new COVID-19 vaccine, composed of two critical antigenic fragments of the S1 and S2 region of severe acute respiratory syndrome coronavirus 2 as well as the whole nucleocapsid protein (N), which was formulated with either alum or alum plus monophosphoryl lipid A (MPLA) adjuvant combinations. METHODS: From within the spike protein S1 region, a fragmented protein P1 (MW:33 kDa) which includes the receptor-binding domain (RBD), another fragment protein P2 (MW:17.6) which contains important antigenic epitopes within the spike protein S2 region, and N protein (MW:46 kDa) were obtained after recombinant expression of the corresponding gene regions in Escherichia coli BL21. For use in immunization studies, three proteins were adsorbed with aluminum hydroxide gel and with the combination of aluminum hydroxide gel plus MPLA. RESULTS: Each of the three protein antigens produced strong reactions in enzyme-linked immunosorbent assays and Western blot analysis studies performed with convalescent COVID-19 patient sera. In mice, these combined protein vaccine candidates elicited high titer anti-P1, anti-P2, and anti-N IgG and IgG2a responses. These also induced highly neutralizing antibodies and elicited significant cell-mediated immunity as demonstrated by enhanced antigen-specific levels of interferon-γ (INF-γ) in the splenocytes of immunized mice. CONCLUSION: The results of this study showed that formulations of the three proteins with Alum or Alum + MPLA are effective in terms of humoral and cellular responses. However, since the Alum + MPLA formulation appears to be superior in Th1 response, this vaccine candidate may be recommended mainly for the elderly and immunocompromised individuals. We also believe that the alum-only formulation will provide great benefits for adults, young adolescents, and children.

Recombinant outer membrane protein Q and putative lipoprotein from Bordetella pertussis inducing strong humoral response were not protective alone in the murine lung colonization model.

Despite high vaccination coverage after introduction of whole cell (wP) and acellular pertussis (aP) vaccines, pertussis resurgence has been reported in many countries. aP vaccines are commonly preferred due to side effects of wP vaccines and formulated with aluminum hydroxide (Alum), which is not an effective adjuvant to eliminate Bordetella pertussis. Low efficiency of current aP vaccines is thought to be the main reason for the resurgence for which newer generation aP vaccines are needed. In the present study, immunogenicity and protective efficacy of outer membrane protein Q (OmpQ) and a putative lipoprotein (Lpp) from B. pertussis were investigated in mice by using two diefrent adjuvants, monophosphoryl lipid A (MPLA) or Alum. OmpQ and putative Lpp were cloned, expressed, and purified from Escherichia coli. The proteins were formulated to immunize mice. Both recombinant OmpQ and putative Lpp induced a significant increase in immunoglobulin G1 (IgG1) and immunoglobulin G2a (IgG2a) responses compared to the control group. Moreover, MPLA-adjuvanted formulations resulted in higher IgG2a levels than Alum-adjuvanted ones. However, there were no significant differences between test and control groups regarding interferon-gamma (IFN-γ) levels, and the mice lung colonization experiments indicated that neither rOmpQ nor rLpp could confer protection alone against B. pertussis challenge.

Immunogenicity and protective efficacy of recombinant iron superoxide dismutase protein from Bordetella pertussis in mice models.

Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. Although availability of effective pertussis vaccines reportedly decreases the incidence of the disease, B. pertussis circulation in populations has not been eliminated. Thus, it is necessary to find new protein candidates with greater immune protective capacities than the currently available acellular pertussis vaccines. In this study, iron superoxide dismutase (FeSOD) gene (sodB) was cloned, expressed in Escherichia coli and recombinant FeSOD protein thence purified. The recombinant protein (rFeSOD) was formulated with aluminum hydroxide (Alum) or monophosphoryl lipid A (MPLA) and injected intraperitoneally to immunize mice, after which IgG1, IgG2a and IFN-γ titers were measured to assess humoral and cellular responses, respectively, to these immunizations. The extent of bacterial colonization in lungs of intranasally challenged mice was determined 5, 8 and 14 days post-challenge. IgG1 and IgG2a responses were significantly stronger in mice that had been immunized with rFeSOD-MPLA than in those that had received rFeSOD-Alum (P < 0.05). Additionally, IgG2a titers were higher in mice vaccinated with recombinant protein FeSOD (rFeSOD) formulated with MPLA, especially after the second immunization. Immunization with rFeSOD-MPLA also provided a modest, but significant decrease in bacterial counts in lungs of mice (P < 0.05). Antigen specific-IFN-γ responses were significantly stronger in the group vaccinated with rFeSOD-MPLA, which could account for the lower bacterial counts. These findings suggest that rFeSOD protein formulated with MPLA has potential as an acellular pertussis vaccine candidate component.

Pertussis vaccines: state-of-the-art and future trends.

Bordetella pertussis is the causative agent of whooping cough (pertussis) which is a worldwide vaccine preventable acute respiratory illness that predominantly involves infants. The reactogenicity of whole-cell (Pw) vaccines and the difficulty of their consistent production have led to the development of acellular pertussis (Pa) vaccines. However, despite high vaccination coverage using either Pw or Pa and introduction of adolescent and adult vaccines with reduced antigen content, there are still reports about the circulation of the microorganism in populations, morbidity in infants and increasing incidence of pertussis among adolescent and adults who transmit the infection to yet unimmunized infants. Waning vaccine-induced immunity and antigenic divergence in circulating strains seem to be the major problems accounting for resurgence of pertussis. Considering the need for new vaccination strategies, improvement of current Pa vaccines by including new virulence factors would probably be the most rationale strategy. Recent advances in B. pertussis proteomics, subproteomics and immunoproteomics greatly aided in identifying novel antigens of the pathogen. Future studies involving quantitative transcriptomic and proteomic profiling of host-B. pertussis interactions, studying gene expression in vivo and reverse vaccinology will also be very promising approaches and tools to develop pertussis vaccines inducing long term immunity.

Immune responses against chimeric DNA and protein vaccines composed of plpEN-OmpH and PlpEC-OmpH from Pasteurella multocida A:3 in mice.

Pasteurella multocida is a pathogenic bacterium causing many diseases that are of significant economic importance to livestock industries. Outer membrane protein H (ompH) gene and two fragments of Pasteurella lipoprotein E (plpE) gene, namely plpEN and plpEC, were cloned from P. multocida A:3. Three DNA vaccine formulations, namely pCMV-ompH, pCMV-plpEN-ompH and pCMV-plpEC-ompH and two protein-based prototype vaccines, alum adjuvanted PlpEN-OmpH and PlpEC-OmpH, were generated. Antibody levels were induced in mice vaccinated with chimeric DNA or protein vaccines. A significant (p < 0.05) increase in serum IFN-g titer was obtained by vaccination with 100 µg of pCMV-ompH, pCMV-plpEC-ompH and PlpEC-OmpH. DNA vaccines did not provide protection upon intraperitoneal challenge with 10 LD50 of live P. multocida A:3. However, 40% protection was conferred by 100 µg of PlpEC-OmpH which was not statistically significant. These results showed that plpEN-ompH and plpEC-ompH chimeric DNA vaccines and alum adjuvanted PlpEN-OmpH or PlpEC-OmpH protein vaccines were immunogenic but not protective against P. multocida A:3 in mice. Prime-boost strategies, i.e. priming with DNA vaccines and boost with protein formulations or different adjuvants can be utilized to obtain significant protection.

Immunogenicity and protective efficacy of the recombinant Pasteurella lipoprotein E and outer membrane protein H from Pasteurella multocida A:3 in mice.

Pasteurella multocida serotype A:3 is a Gram-negative bacterial pathogen, one of the causative agents of shipping fever of cattle. In this study, outer membrane protein H (ompH) and Pasteurella lipoprotein E (plpE) genes were cloned and plpEC-ompH fusion was constructed and expressed in Escherichia coli. Recombinant PlpE, OmpH and PlpEC-OmpH fusion proteins were purified and formulated with oil-based and oil-based CpG ODN adjuvants. Antibody responses in mice vaccinated with recombinant PlpE and PlpEC-OmpH proteins formulated with both adjuvants were significantly (p<0.05) increased. However, a significant (p<0.05) increment in serum IFN-γ level was only observed upon immunization with oil-based CpG formulations. Protectivity of the vaccines were evaluated via intraperitoneal challenge of mice with 10 LD50 of P. multocida A:3. The recombinant proteins PlpE and PlpEC-OmpH fusion conferred 100% protection when formulated with oil-based CpG ODN while the protectivity was found to be 80% and 60%, respectively when only oil-based adjuvant was used in respective formulations. These findings indicated that the recombinant PlpE or PlpEC-OmpH fusion proteins formulated with oil-based CpG ODN adjuvant are possible acellular vaccine candidates against shipping fever.

A comprehensive analysis of Bordetella pertussis surface proteome and identification of new immunogenic proteins.

Whooping cough, caused by the gram negative pathogen Bordetella pertussis, is a worldwide acute respiratory disease that predominantly involves infants. In the present study, surface proteins of B. pertussis Tohama I and Saadet strains were identified by using 2DE followed by MALDI-TOF-MS/MS analysis and also geLC-MS/MS. With these approaches it was possible to identify 45 and 226 proteins, respectively. When surface proteins of the strains were separated by 2DE and analyzed by Western blotting for their reactivity, a total of 27 immunogenic spots which correspond to 11 different gene products were determined. Glutamine-binding periplasmic protein, leu/ile/val-binding protein, one putative exported protein, and iron-superoxide dismutase (Fe-SOD) were found as immunogenic for the first time in Bordetella. Of a total of 226 proteins identified, 16 were differentially expressed in B. pertussis Saadet and Tohama I strains. Five proteins were expressed only in Saadet (adhesin, chaperone protein DnaJ, fimbrial protein FimX, putative secreted protein Bsp22 and putative universal stress protein), and two (ABC transporter substrate-binding protein and a putative binding protein-dependent transport periplasmic protein) only in Tohama I.

In vitro and in vivo evaluations of PLGA microsphere vaccine formulations containing pDNA coexpressing hepatitis B surface antigen and Interleukin-2.

PURPOSE: DNA-based vaccines encoding viral antigens have been shown to elicit immune responses in animal models. In this study, a plasmid DNA (pDNA) coexpressing the middle envelope protein of hepatitis B virus (HBV) and Interleukin-2 (IL-2) was incorporated into Poly (D,L-lactic-co-glycolic acid) (PLGA) microspheres and three different formulations were investigated for their potential as a vaccine delivery system. METHODS: Emulsion solvent evaporation methods of water-in-oil-in-water (w/o/w) and oil-in-water (o/w) were used to generate three different formulations in which PLGA microspheres contained pDNA either encapsulated within or adsorbed onto the microspheres. RESULTS: In vaccine formulation A2, prepared using the (w/o/w) method, pDNA was encapsulated within the microspheres. The other two formulations (B2 and B2a) were prepared using the (o/w) method and B2 contained pDNAs encapsulated within the microspheres while B2a contained pDNAs adsorbed onto the microspheres. pDNA loading efficiencies of A2, B2 and B2a were determined to be 15%, 25% and 45%, respectively. In vitro release of pDNAs from microspheres was evaluated for a 45-day period with no conformational changes and A2 displayed slower release than that of the B2 and B2a. When mice were immunized from anterior tibialis muscle using A2, B2 and B2a formulations containing 100 microg pDNA, antibody responses were detected for 6 months in mice sera. CONCLUSIONS: Although all PLGA microsphere formulations containing pDNA elicited antibody responses by the end of the 6th month, the antibody titers were found to be higher with B2 and B2a formulations in comparison to A2 formulation and the naked pDNA in saline.

Immunoproteomic analysis of Bordetella pertussis and identification of new immunogenic proteins.

Bordetella pertussis is the causative agent of highly communicable respiratory infection whooping cough (pertussis) which remains one of the world's leading causes of vaccine-preventable deaths. In the present study, total soluble proteins extracted from two B. pertussis strains, Tohama I and the local isolate Saadet were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the antisera obtained from the mice immunized with inactivated whole cells as well as those collected from the mice challenged intraperitoneally with live cells of each strain. Of a total of 25 immunogenic proteins identified, 21 were shown to be the novel antigens for B. pertussis.

[Development studies of lyophilized standard diphtheria-tetanus-pertussis vaccines]. / Liyofilize standart difteri-tetanoz-bogmaca asilari gelistirme çalismalari.

In this study, diphtheria, tetanus and pertussis vaccine components were prepared as the formulations of diphtheria-tetanus-pertussis (DTP), diphtheria-tetanus (DT) for children, diphtheria-tetanus (Td) for adults, and tetanus toxoid (TT), respectively. Alhydrogel-adsorbed vaccines prepared to contain the stabilizing substances were lyophilized and the immunogenicity tests were carried out both in vivo and in vitro. The potencies of the tetanus component of the vaccines were obtained by the lethal challenge test in mice. The values were found as 144.86 IU/ml for lyophilized adsorbed (LA)-DTP, 116.5 IU/ml for LA-DT, 98.25 IU/ml for LA-Td and 96.2 IU/ml for LA-TT. Anti-tetanus IgG and anti-diphteria IgG levels determined by ELISA method were found high in the sera taken from the mice immunized with the above-mentioned vaccines. Anti-B.pertussis fimbria IgG antibody levels were also high by both ELISA and microagglutination tests. The test preparations were then compared to adsorbed liquid vaccines and it was shown that the components were quite stable in the lyophilized formulations. It was concluded that the formulations prepared in this study can be used as standard vaccines after being calibrated against World Health Organization standards.

[Rapid purification of Bordetella pertussis fimbria 2, 3 proteins and their immunogenicities]. / Bordetella pertussis fimbriya 2, 3 proteinlerinin hizli saflastirilmasi ve immunojenitesi.

Bordetella pertussis fimbria antigens play an important role in the adhesion to the cell surfaces, and induce the formation of protective antibodies in the host. The aim of this study was to purify the fimbria proteins by a rapid and easy method which involved Superdex 75 gel filtration chromatography, and to investigate their immunogenicity. The fimbria proteins purified from the different strains showed single protein bands in 12.5% SDS-PAGE (Sodium dodecyl sulphate-polyacrylamide gel electrophoresis) method. Depending on the strain used, the proteins were observed as Fim2 and Fim3 subunits or only Fim 2 subunit on 16% SDS-PAGE. The purified subunits were confirmed by Western and dot blotting analysis with monoclonal antibodies BP F2 (anti-Fim2) and BPC10 (anti-Fim 3). The immunogenicity studies performed in the mice revealed that purified fimbria proteins have led to higher agglutinin response than the vaccine with whole cell pertussis antigens. It was determined that the extent of agglutinin responses were different for different strains from which the fimbria proteins were purified. In addition, each B. pertussis strain showed a different agglutination reaction with different anti-Fim sera. The results, overall, pointed out the significance of fimbria structure of the B. pertussis strain used for vaccine manufacture and control.

Rapid purification of pertussis toxin (PT) and filamentous hemagglutinin (FHA) by cation-exchange chromatography.

Pertussis toxin (PT) and filamentous hemagglutinin (FHA) were purified from culture supernatant of Bordetella pertussis Saadet and Tohama strains, using CM-Sepharose CL-6B cation-exchange chromatography. By the rapid purification method described here, both proteins were separately eluted from the same column in pure forms. The PT and FHA in the extract of culture supernatant were bounded to CM-Sepharose CL-6B cation-exchange column in 50 mM phosphate buffer containing 2 M urea (Buffer A), pH 6.0. Then the PT was eluted from the column with Buffer A (pH 7.4) and after elution of the PT, the FHA was eluted with 0.5 M NaCl in 50 mM phosphate buffer. Pertussis toxin and filamentous haemagglutinin purified by this procedure were electrophoretically and immunologically identical to the reference preparations.