Development and In Vivo Evaluation of Recombinant Multi-Epitope Vaccine (ABOR) with Chitin Microparticles as Adjuvant Against Brucella Abortus

Intranasal administration of adjuvant-combined recombinant influenza virus HA vaccine protects mice from the lethal H5N1 virus infection

BackgroundInfluenza A virus is one of the leading causes of annual mortality. The emerging of novel escape variants of the influenza A virus is still a considerable challenge in the annual process of vaccine production. The evolution of vaccines ranks among the most critical successes in medicine and has eradicated numerous infectious diseases. Recently, multi-epitope vaccines, which are based on the selection of epitopes, have been increasingly investigated.ResultsThis study utilized an immunoinformatic approach to design a recombinant multi-epitope vaccine based on a highly conserved epitope of hemagglutinin, neuraminidase, and membrane matrix proteins with fewer changes or mutate over time. The potential B cells, cytotoxic T lymphocytes (CTL), and CD4 T cell epitopes were identified. The recombinant multi-epitope vaccine was designed using specific linkers and a proper adjuvant. Moreover, some bioinformatics online servers and datasets were used to evaluate the immunogenicity and chemical properties of selected epitopes. In addition, Universal Immune System Simulator (UISS) in silico trial computational framework was run after influenza exposure and recombinant multi-epitope vaccine administration, showing a good immune response in terms of immunoglobulins of class G (IgG), T Helper 1 cells (TH1), epithelial cells (EP) and interferon gamma (IFN-g) levels. Furthermore, after a reverse translation (i.e., convertion of amino acid sequence to nucleotide one) and codon optimization phase, the optimized sequence was placed between the two EcoRV/MscI restriction sites in the PET32a+ vector.ConclusionsThe proposed “Recombinant multi-epitope vaccine” was predicted with unique and acceptable immunological properties. This recombinant multi-epitope vaccine can be successfully expressed in the prokaryotic system and accepted for immunogenicity studies against the influenza virus at the in silico level. The multi-epitope vaccine was then tested with the Universal Immune System Simulator (UISS) in silico trial platform. It revealed slight immune protection against the influenza virus, shedding the light that a multistep bioinformatics approach including molecular and cellular level is mandatory to avoid inappropriate vaccine efficacy predictions.

To perform prokaryotic expression and preliminary characterization of the recombinant poly-epitope vaccine EgG1Y162-2 (4) against cystic echinococcosis. The recombinant poly-epitope vaccine EgG1Y162-2 (4) against Echinococcus granulosus based on the linker GSGGSG was subjected to structural three-dimensional (3D) modeling using immunoinformatics to analyze the structural changes and evaluate the antigenicity of the vaccine. The pET30a-EgG1Y162-2 (4) recombinant plasmid was generated using double digestion with EcoR I and Sal I, and then transformed into competent cells. Following protein induction with isopropyl-β-D-thiogalactoside (IPTG), the prokaryotic expression proteins were characterized using Western blotting, and the antigenicity of the recombinant protein was analyzed using sera from cystic echinococcosis patients and health volunteers. The four EgG1Y162-2 proteins coupled by the 3D structure of the recombinant vaccine EgG1Y162-2 (4) presented independent and effective expression and good antigenicity. The highest protein expression was detected in the supernatant following induction of the recombinant plasmid pET30a-EgG1Y162-2 (4) by 0.2 mmol/L IPTG at 37 °C for 4 h, and a pure protein component was seen following elution with 60 mmol/L imidazole. Western blotting analysis of the recombinant multiepitope protein HIS-EgG1Y162-2 (4) showed a band at approximately 39 kDa, and this band was recognized by sera from cystic echinococcosis patients. A recombinant poly-epitope vaccine EgG1Y162-2 (4) against cystic echinococcosis has been successfully constructed, which provides a preliminary basis for researches on recombinant multi-epitope vaccine against cystic echinococcosis.

In recent years, elk (Cervus canadensis) have been implicated as the source of Brucella abortus infection for numerous cattle herds in the Greater Yellowstone Area. In the face of environmental and ecological changes on the landscape, the range of infected elk is expanding. Consequently, the development of effective disease management strategies for wild elk herds is of utmost importance, not only for the prevention of reintroduction of brucellosis to cattle, but also for the overall health of the Greater Yellowstone Area elk populations. In two studies, we evaluated the efficacy of B. abortus strain RB51 over-expressing superoxide dismutase and glycosyltransferase for protecting elk from infection and disease caused by B. abortus after experimental infection with a virulent B. abortus strain. Our data indicate that the recombinant vaccine does not protect elk against brucellosis. Further, work is needed for development of an effective brucellosis vaccine for use in elk.

Brucellosis is a bacterial zoonotic disease affecting several mammalian species that is transmitted to humans by direct or indirect contact with infected animals or their products. In cattle, brucellosis is almost invariably caused by Brucella abortus. Live, attenuated Brucella vaccines are commonly used to prevent illness in cattle, but can cause abortions in pregnant animals. It is, therefore, desirable to design an effective and safer vaccine against Brucella. We have used specific Brucella antigens that induce immunity and protection against B. abortus. A novel recombinant multi-epitope DNA vaccine specific for brucellosis was developed. To design the vaccine construct, we employed bioinformatics tools to predict epitopes present in Cu–Zn superoxide dismutase and in the open reading frames of the genomic island-3 (BAB1_0260, BAB1_0270, BAB1_0273, and BAB1_0278) of Brucella. We successfully designed a multi-epitope DNA plasmid vaccine chimera that encodes and expresses 21 epitopes. This DNA vaccine induced a specific humoral and cellular immune response in BALB/c mice. It induced a typical T-helper 1 response, eliciting production of immunoglobulin G2a and IFN-γ particularly associated with the Th1 cell subset of CD4+ T cells. The production of IL-4, an indicator of Th2 activation, was not detected in splenocytes. Therefore, it is reasonable to suggest that the vaccine induced a predominantly Th1 response. The vaccine induced a statistically significant level of protection in BALB/c mice when challenged with B. abortus 2308. This is the first use of an in silico strategy to a design a multi-epitope DNA vaccine against B. abortus.

Brucella abortus causes brucellosis in humans and cows worldwide. The lack of effective treatment against zoonotic infection brucellosis in cows necessitates finding an urgent remedy. Therefore, the research aims to create a multi-epitope vaccination to combat infectious brucellosis. An in silico immunoinformatic approach was employed using bioinformatics tools and databases to design multimeric subunit vaccination, as opposed to generating the vaccine from the entire pathogen. Essential antigenic proteins, Biotin synthase (bioB) and Nitrous oxide reductase (nosZ), were identified as non-toxic, non-allergenic, and stable through toxicity, allergenicity, and physicochemical analyses. Secondary structures of immunogenic proteins were predicted, followed by the prediction and evaluation of their tertiary structures. Subsequently, shortlisted proteins underwent (T and B) cell epitope prediction. Highly conserved antigenic, immunogenic, nonal¬lergic, and nontoxic epitopes have been short-listed for vaccination advancement. Multi-epitope vaccine (comprising 481 amino acids) constructed with (shortlisted 33 epitopes, linkers, EAAAK, AAY, KK, adju¬vant, beta-defensin three). Moreover, structure prediction with model evaluation was performed to evaluate structural stability and quality. Additionally, the docking technique was employed to predict the binding potential of Toll-like receptor nine. Dynamics simulation predicted the structural flexibility of the docked complex to assess its reliability. The vaccine showed immunological effects, indicating promising results. However, experiments and laboratory research are necessary to prove the vaccine’s immunogenicity.

A novel multi-epitope-based peptide recombinant influenza A vaccine prototype utilizing neuraminidase and hemagglutinin surface proteins: From in silico to preliminary study.

Humoral immune response of BALB/c mice to a vaccinia virus recombinant expressing Brucella abortus GroEL does not correlate with protection against a B. abortus challenge

Brucella is a common kind of bacteria that has the ability to live within cells and may cause diseases that can be transmitted between animals and humans. Current medical therapy struggles to effectively eradicate Brucella. Thus, it is necessary to develop a multi-epitope vaccine (MEV) in order to effectively prevent Brucella infection. To achieve this objective, we used the reverse vaccinology methodology based on omp19 and Bacterial surface antigen (D15). After conducting our research, we successfully identified 2 cytotoxic T lymphocyte (CTL) epitopes, 2 helper T lymphocyte (HTL) epitopes, and 2 linear B cell epitopes from Omp19 and Bacterial surface antigen (D15). These epitopes will be further examined in our study. In order to maintain the proper folding of the protein, we connected GGGS and EAAAK consecutively. Adjuvants are added to the N-terminal of the vaccine peptide to boost its immunogenicity. In order to assess the immunity, stability, protection, and practicality of the final MEV, a construct consisting of 387 amino acids was created by connecting linkers and adjuvants. Furthermore, molecular docking and simulations using molecular dynamics were conducted to confirm the binding strength and durability of the MEV-TLR5. Subsequently, codon adaptation and in silico cloning analyses were conducted to determine the potential codons for expressing the MEV. The findings indicated that the MEV exhibited a significant level of immunogenicity. This work has collectively established a theoretical foundation for the development of a vaccine against Brucella.

Brucella abortus vaccines help control bovine brucellosis. The RB51 strain is a live attenuated vaccine with low side effects compared with other live attenuated brucellosis vaccines, but it provides insufficient protective efficacy. Cell-mediated immune responses are critical in resistance against intracellular bacterial infections. Therefore, we hypothesized that the listeriolysin O (LLO) expression of Listeria monocytogenes, BAX, and SMAC apoptotic proteins in strain RB51 could enhance vaccine efficacy and safety. B. abortus RB51 was transformed separately with two broad-host-range plasmids (pbbr1ori-LLO and pBlu–mLLO-BAX-SMAC) constructed from our recent work. pbbr1ori-LLO contains LLO, and pBlu–mLLO-BAX-SMAC contains the mutant LLO and BAX-SMAC fusion gene. The murine macrophage-like cell line J774A.1 was infected with the RB51 recombinant strain containing pBlu-mLLO-BAX-SMAC, RB51 recombinant strain containing LLO, and RB51 strain. The bacterial cytotoxicity and survival and apoptosis of host cells contaminated with our two strain types—RB51 recombinants or the parental RB51—were assessed. Strain RB51 expressing mLLO and BAX-SMAC was tested in BALB/c mice and a cell line for enhanced modulation of IFN-γ production. LDH analysis showed that the RB51-mLLO-BAX-SMAC and RB51-LLO strains expressed higher cytotoxicity in J774A.1 cells than RB51. In addition, RB51 recombinants had lower macrophage survival rates and caused higher levels of apoptosis and necrosis. Mice vaccinated with the RB51 recombinant containing mLLO-BAX-SMAC showed an enhanced Th1 immune response. This enhanced immune response is primarily due to bacterial endosome escape and bacterial antigens, leading to improved apoptosis and cross-priming. This potentially enhanced TCD8+- and T cell-mediated immunity leads to the increased safety and potency of the RB51 recombinant (RB51 mLLO-BAX-SMAC) as a vaccine candidate against B. abortus.

Brucella abortus RB51: enhancing vaccine efficacy and developing multivalent vaccines

Enhanced efficacy of recombinant Brucella abortus RB51 vaccines against B. melitensis infection in mice

Background: Live attenuated Brucella abortus strains 19 and RB51 vaccines have been used as a key method for the control and eradication of brucellosis in cattle worldwide for decades. Due to certain limitations of these live vaccines, research has been undertaken for the development of an ideal more effective and safer vaccine for animals and human brucellosis. Objective: The main objective of this study was to compare the humoral immune responses (HIR) between the heat-inactivated Brucella abortus biovar 3 and attenuated live RB51 vaccines in native cattle of Bangladesh. Materials and Methods: The methods of isolation, identification, preparation of inoculum dose (10 × 1010 cfu/5 ml) and heat inactivation of B. abortus biovar 3 was followed as described earlier. Each of the three B. abortus sero-negative native cows was inoculated with heat-inactivated B. abortus vaccine @ 5.0 ml (10 × 1010 cfu /5 ml)/ cow SC single injection. Similarly, each of five native calves of 6 to 9 months old was inoculated with live attenuated RB51 vaccine (CZ Veterinaria, SA, Spain) @ 2.0 ml (10-34×109) SC as single dose. The sera of cows were collected at 0, 7, 14, 21, 28, 40, 60 and 90 days post vaccination, whereas the sera of the calves were collected at 0, 7, 14, 21, 28, 60, 90, 120, 150 and 180 days post-vaccination. All the collected sera of both the groups were tested to evaluate antibody titer by RBT followed by ELISA with commercial tests kits. Results: The HIR of the cows inoculated with heat-inactivated vaccine showed antibody (Ab) titer started to rise significantly (p < 0.05) from the 14 days (OD 0.2116 ± 0.0397, Ab titer 1:120) and reached a peak level at 28 days (OD 0.319 ± 0.172, Ab titer 1:800) and then started to decline significantly (p < 0.05) from 40 days (OD 0.234 ± 0.0415, Ab titer 1:35) to 60 days (OD 0.094 ± 0.0075, Ab titer 0). The mean Ab titer in calves inoculated with RB51 vaccine showed that Ab titer started to appears insignificantly (p ˃ 0.05) from day 7 (OD 0.094 ± 0.01603) and reached peak level at day 60 days (OD 0.592 ± 0.398), changes are very significant from day 0 (p < 0.05), after 60 days Ab level start to decrease and reach at lowest level at day 150 (OD 0.112 ± 0.0188), Ab level found similar to day 0 (OD 0.0826 ± 0.00517) at 180 days (OD 0.0822 ± 0.00249). Conclusions: The S19 and RB51 are the approved B. abortus vaccine strains have been widely and successfully used with some limitations to prevent bovine brucellosis worldwide. In addition to live attenuated and inactivated vaccines, recombinant genes, proteins, vectors, DNA and recombinant mutant vaccines have also been evaluated for the prevention of brucellosis but further research would be required to develop an ideal vaccine for both the humans and animals.

Over-expression of homologous antigens in a leucine auxotroph of Brucella abortus strain RB51 protects mice against a virulent B. suis challenge

Brucella spp. are a bacterium that cause brucellosis, a zoonotic disease, which is commonly seen in cattle, sheep, goats, swine, and canines. Brucellosis is a problem worldwide, although it is eradicated in some countries (Garin-Bastuji et al. 1998). The reason for designing recombinant DNA (rDNA) vaccines opposed to utilizing the live-attenuated vaccines on the market is that they cannot be given to pregnant animals without potentially causing abortion, while an rDNA vaccine should be safe for pregnant animals since it does not contain viable bacteria. Also, there are no serological tests that can accurately distinguish between an animal vaccinated with the current vaccines available for brucellosis and an animal with an active Brucella infection because the current vaccines are live-attenuated and interfere with diagnostics. Recombinant DNA vaccines eliminate many safety concerns, which is why two rDNA vaccines were constructed in the same way, except for the length of the immunogenic epitopes selected from Brucella abortus. The epitopes from B. abortus that were chosen are present in the other main species of Brucella because the main species of Brucella, such as B. abortus, B. melitensis, and B. suis, are 99% identical at the DNA level. The epitopes range in length from 25 to 70 amino acids. Each vaccine contained a specific insert, with different lengths of epitopes and an antibiotic resistance gene that was incorporated for selection of successfully transfected cells during in vitro testing. After the vaccines were constructed, they were tested in vitro to determine the vaccine’s ability to enter different cell types and show protein expression in a variety of cells. The vaccines were tested in a caprine model to determine the vaccine’s ability to be successfully expressed in mammalian cells in vivo. It was determined that no significant antibody concentration was detected via the testing methods utilized.