Development Of Polyvalent Vaccine Research Articles

New Engineered-Chimeric Botulinum Neurotoxin Mutant Acts as an Effective Bivalent Vaccine Against Botulinum Neurotoxin Serotype A and E.

Botulinum neurotoxins (BoNTs), including serotypes A and E, are potent biotoxins known to cause human poisoning. In addition to the critical protective antigen found in the full BoNT molecule, the receptor binding domain (Hc domain), BoNTs also harbour another essential protective antigen-the light chain-translocation domain (L-HN domain). Leveraging these pivotal protective antigens, we genetically engineered a series of inactivated chimeric molecules incorporating L-HN and Hc domains of BoNT/A and E. The structure of these chimeric molecules, mirror BoNT/A and E, but are devoid of enzyme activity. Experimental findings demonstrated that a lead candidate mEL-HN-mAHc harnessing the inactivated protease LCHN/E with the mutated gangliosides binding site Hc/A (mE-mA) elicited robust immune protection against BoNT/A and E simultaneously in a mouse model, requiring low immune dosages and minimal immunisations. Moreover, mE-mA exhibited high protective efficacy against BoNT/A and E in guinea pigs and New Zealand white rabbits, resulting in elevated neutralising antibody titres. Furthermore, mE-mA proved to be a more stable and safer vaccine compared to formaldehyde-inactivated toxoid. Our data underscore the genetically engineered mE-mA as a highly effective bivalent vaccine against BoNT/A and E, paving the way for the development of polyvalent vaccines against biotoxins.

Nucleic acid-based vaccine platforms against the coronavirus disease 19 (COVID-19).

The coronavirus disease 2019 (COVID-19) pandemic has infected 673,010,496 patients and caused the death of 6,854,959 cases globally until today. Enormous efforts have been made to develop fundamentally different COVID-19 vaccine platforms. Nucleic acid-based vaccines consisting of mRNA and DNA vaccines (third-generation vaccines) have been promising in terms of rapid and convenient production and efficient provocation of immune responses against the COVID-19. Several DNA-based (ZyCoV-D, INO-4800, AG0302-COVID19, and GX-19N) and mRNA-based (BNT162b2, mRNA-1273, and ARCoV) approved vaccine platforms have been utilized for the COVID-19 prevention. mRNA vaccines are at the forefront of all platforms for COVID-19 prevention. However, these vaccines have lower stability, while DNA vaccines are needed with higher doses to stimulate the immune responses. Intracellular delivery of nucleic acid-based vaccines and their adverse events needs further research. Considering re-emergence of the COVID-19 variants of concern, vaccine reassessment and the development of polyvalent vaccines, or pan-coronavirus strategies, is essential for effective infection prevention.

Precise Assembly of Multiple Antigens on Nanoparticles with Specially Designed Affinity Peptides.

Antigen proteins, assembled on nanoparticles, can be recognized by antigen-presenting cells effectively to enhance antigen immunogenicity. The ability to simultaneously display multiantigens on the same nanoparticle could have numerous applications but remained technical challenges. Here, we described a method for precise assembly of multiple antigens on nanoparticles with specially designed affinity peptides. First, we designed and screened affinity peptides with high affinity and specificity, which could respectively target the key amino acid residues of classical swine fever virus (CSFV) E2 protein or porcine circovirus type 2 capsid protein (PCV2 Cap) accurately. Then, we conjugated the antigen proteins to poly(lactic acid-glycolic acid) copolymer (PLGA) and Gram-positive enhancer matrix (GEM) nanoparticles through the peptides and perfectly assembled two kinds of multiantigen display nanoparticles with different particle sizes. Subsequently, the immunological properties of the assembled nanoparticles were tested. The results showed that the antigen display nanoparticles could promote the maturation, phagocytosis, and proinflammatory effects of antigen-presenting cells (APCs). Besides, compared with the antigen proteins, multiantigen display nanoparticles could induce much higher levels of antibodies and neutralizing antibodies in mice. This strategy may provide a technical support for the study of protein structure and the research and development of polyvalent vaccines.

Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens.

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

Sero-prevalence of foot and mouth disease in cattle in Borena Zone, Oromia regional state, Ethiopia

ObjectiveTo determine the sero-prevalnce of FMD and indicate patterns of animal movement in Borena zone, Ethiopia.IntroductionThe Foot and mouth disease (FMD) virus is a highly contagious and economically devastating trans boundery disease of cloven-hooved domestic and wild animals1.MethodsA cross-sectional study was carried out between April and November 2015 to investigate the sero-prevalence of foot-and-mouth disease (FMD) in cattle using serology and questionnaire servey in Borena zone.ResultsA total of 363 sera samples were collected from nine peasant associations found in three different districts. An overall seroprevalence of 42.7% (95%: CI= 37.7-47.84) was found during the study. There was statistically significant difference among the districts (χ2 = 10.43, p=0.005) and the highest prevalence was found in Dire district which accounted for 52.8% (95%: CI, 44.0-61.4). Soda peasant association of Dire district and Surupa peasant association of Yabello district accounted for highest sero-prevalence 65.5% (95%: CI, 49.4-78.5) and 65.0% (95%: CI= 40.4-78.5), respectively. Statistical significant difference in footand-mouth disease seroprevalence (χ2 =31.1, p=0.000) was found among the peasant associations. Similarly, there was significance difference (χ2 =17.4, p=0.000) in the prevalence of foot-and-mouth disease between age groups. Though the seroprevalence foot-and-mouth disease was higher in females than in males, there was no significant difference (χ2=1.63, p=0.202) between sex. The different risk factors analyzed during this study indicated that, peasant associations (PAs), district and age were seen to be significantly associated (p<0.05) with the seroprevalence of foot-and-mouth disease. The questionnaire survey revealed that foot-and-mouth disease outbreak was commonly seen during June to August (Short rainy season) and December to February (Long dry season), locally called Adolessa and Bona, respectively. Younger (1-3 years) animals were most susceptible than calf and adults (>3years). Moreover, an extrinsic factor like dry season enforces pastoralist to travel a longer distance to look for grazing lands and water sources that creates suitable conditions for foot-and-mouth disease transmission between infected and susceptible animals.ConclusionsFMD is an important transboundery animal disease that affects the livelihood of farmers and economy of the country. In pastoral areas like Borena where livestock movement is common during dry season, the disease is devastating and spreading from one area to the other. Therefore, an extensive regular serological survey, virus isolation, and characterizations of FMD virus need to be conducted for a possible development of poly-valent vaccines that contains commonly circulating serotypes of FMD virus in Ethiopia.

Six genes of ompA family shuffling for development of polyvalent vaccines against Vibrio alginolyticus and Edwardsiella tarda

Polyvalent vaccines against more than one species of pathogens are especially important due to the complex ecosystem in aquaculture. We have previously shown that the development of polyvalent vaccines by shuffling six ompA genes from different bacteria with V. parahaemolyticus VP0764 primers. Here, we used the same 6 genes, V. alginolyticus VA0764 and VA1186, V. parahaemolyticus VP0764 and VP1186, E. tarda ompA and E. coli ompA, but with E. tarda ompA primers to develop new polyvalent vaccines. By this approach, we identified 7 potential polyvalent vaccines that were effective against both V. alginolyticus and E. tarda infections. Furthermore, the innate immunity triggered by the vaccines were also explored in three groups, no protection (group I), protection against V. alginolyticus (group II), and protection against both V. alginolyticus and E. tarda (group III). The transcription of IL-1β, IL-6, IL-8, C3b and NF-kB were significantly increased in group II and group III but not group I, where the expression level of group III was higher than group II. In addition, differential activities of succinate dehydrogenase were detected among the three groups. These results indicate the expansion of polyvalent vaccine reservoir with the same shuffling genes but different primers, and promote the understanding of the mechanisms of polyvalent vaccines based on vaccine-induced innate immunity.

Construction and immune protection evaluation of recombinant polyvalent OmpAs derived from genetically divergent ompA by DNA shuffling.

A wide variety of bacterial infections is a major challenge in aquaculture. Development of polyvalent vaccines that can fight against as many pathogens as possible is especially necessary. The present study uses DNA shuffling to create a new hybrid OmpA with improved cross-protection against Vibrio alginolyticus and Edwardsiella tarda through the recombination of six OmpA genes from Vibrio parahaemolyticus, V. alginolyticus, E. tarda and Escherichia coli. Out of the 43 recombinant chimeras genes constructed using VA0764 primers, EompAs-19 was demonstrated as an ideal polyvalent vaccine against infections caused V.alginolyticus and E.tarda. Compared with VA0764, OmpAs-19 had three mutations, which may be a molecular basis of EompAs-19 as an efficient polyvalent vaccine against both V.alginolyticus and E.tarda infections. These results develop a polyvalent vaccine that prevents the infections caused by extracellular and intracellular bacteria. Thus, the present study highlights the way to develop polyvalent vaccines against microbial infections by DNA shuffling.

Synthesis and immunogenicity evaluating of a discontinuous B-cell epitope predicted from influenza virus A/H5N1 HA antigen

To develop vaccine with stable efficiency against easily transforming virus such as influenza A/H5N1 virus, bioinformatic tools have been used to predict conserved epitopes from viral antigens to be used as materials for the development of polyvalent vaccine against this virus. Using this approach, we have successfully predicted one discontinuous B-cell epitope, a peptide of 14 aminoacids on conserved domains of HA antigen from H5N1 influenza A virus. In this study, we report results of the preparation of this discontinuous B-cell epitope as the recombinant fusion form with H:1,2 flagellin from Salmonella typhimurium to increase the immunogenicity of this epitope using genetic manipulating techniques with Escherichia coli as host cell. The immunogenicity of the chemically synthesized predicted epitope and the recombinant epitope was examined by immunization of each of these epitopes to mice. Using hemagglutination inhibition (HI) method, we successfully demonstrated that both anti-sera from mice immunized either with chemically synthesized peptide or with recombinant flagellin H:1,2 fused epitope could recognize and inhibit the agglutination of inactivated influenza H5N1 virus strains, such as A/H5N1/ Chicken13/ Vietnam/LA/2006 and A/Vietnam/CL26/2004 (H5N1) isolated from infected chicken and human in Vietnam.

Cancer–Testis Genes as Candidates for Immunotherapy in Breast Cancer

Cancer-testis (CT) antigens are tumor-associated antigens attracting immunologists for their possible application in the immunotherapy of cancer. Several clinical trials have assessed their therapeutic potentials in cancer patients. Breast cancers, especially triple-negative cancers are among those with significant expression of CT genes. Identification of CT genes with high expression in cancer patients is the prerequisite for any immunotherapeutic approach. CT genes have gained attention not only for immunotherapy of cancer patients, but also for immunoprevention in high-risk individuals. Many CT genes have proved to be immunogenic in breast cancer patients suggesting the basis for the development of polyvalent vaccines.

Protective properties of recombinant IgA1 protease from meningococcus

The study of enzymatic and protective properties of recombinant IgA1 protease in active and mutant form showed that active form of IgA1 protease exhibited species - and type-specificity for mouse and human immunoglobulins. Mutant form, which did not exhibit enzymatic activity, had protective properties against meningococcal infection, induced by meningococcus serogroup A, B and C protecting the mice from lethal infection by living virulent culture of heterologous serogroups of meningococcus. Obtained results make it possible to consider IgA1 protease as a perspective preparation at the stages of development of polyvalent vaccine for protection the people from meningococcal infection of various etiology.

In silico analysis of chimeric recombinant immunogen against three diarrhea causing bacteria

Shigella and Escherichia belong to the Enterobacteriaceae family which are the cause for most of the diarrheal cases in the world. Shigella can cause bacterial dysenteries and shigellosis. One of the most effective proteins for pathogenesis is invasion plasmid antigen C (IpaC). Other bacteria like Enterotoxogenic (ETEC), Enterohemorrhagic (EHEC), and E.coli can also cause diarrhea and produce intestinal disorders. Colonization factor antigen I (CFA/I), a critical virulence protein for these infections, has two subunits i.e. CfaB and CfaE. EHEC Attachment of bacteria is the main step of infection with intimin playing the key role in this function. This study was designed to elicit protection against the majority of diarrheal pathogens via development of polyvalent vaccine against Shigella, ETEC and EHEC. In silico techniques are as best tools to design new vaccines. For this purpose the immunogenic epitopes of CfaB, IpaC and Intimin were identified through bioinformatic tools and were then selected as major antigens to construct a chimeric protein (CII). The humoral and cellular immunities were analyzed bioinformatically. Prediction of allergens and mapping of IgE epitopes were carried out. The bioinformatic analysis showed each domain was folded separately in fusion structure. CII had many T and B cell epitopes in both linear and three-dimensional structures. This prediction of the chimeric construct had the potential to induce CD4+ and CD8+ immune responses against these pathogens. In addition CII could be accessible to surveillance by the immune system in mouse and human. In conclusion, in silico analysis showed that this chimeric protein can be used as a vaccine against Shigella, ETEC and EHEC simultaneously.

Immunogenicity in poultry of in silico predicted - continuous B-cell epitopes from influenza virus H5N1

In order to develop vaccine with stable efficiency against easily transforming virus such as influenza H5N1 virus, bioinformatic tools were used to predict conserved epitopes from viral antigens to be used as materials for the development of polyvalent vaccine against this virus. Using this approach, we have successfully predicted B-cell continuous and discontinuous epitopes on conserved domains of HA and NA antigens from H5N1 influenza A virus. To confirm the immunogenicity of these epitopes, genetic manipulating techniques have been employed, to prepare the recombinant epitopes in E. coli as a fusion form with H:1,2 flagellin antigen from Salmonella Typhimurium and with glutathione S-transferase. These recombinant antigens have been collected, purified and used for animal immunizing. This study shows the results in specific immunogenicity of recombinant B-cell epitopes continuous in chickens. Using HI test (Hemagglutination Inhibition Test), we could successfully prove that the antiserum from both of chicken groups immunized with GST-H:1,2-HeBc and GST-H:1,2-NeBc had specific antibodies could inhibit the agglutination of antigens derived from an influenza H5N1 virus strain isolated from infected chickens in Vietnam. The HI titer of anti-GST-H:1,2-NeBc antibodies was 113,00, that is 1,19 times higher than the HI titer of anti-GST-H:1,2-HeBc antibodies (95,00), while the HI titer of antibodies from chickens immunized with commercial inactivated vaccine H5N1 reached 291,58.

SYNTHESIS AND IMMUNOGENICITY TESTING OF A BIOINFORMATICSPREDICTED B-CELL EPITOPE ON THE HA ANTIGEN FROM INFLUENZA H5N1 VIRUS

In order to develop vaccine with stable efficiency against easily transforming virus such as influenza H5N1 virus, bioinformatic tools were used to predict conserved epitopes from viral antigens to be used as materials for the development of polyvalent vaccine against this virus. Using this approach, we have successfully predicted one B-cell continuous epitope consisting of 21 amino acids on conserved domains of HA antigen from H5N1 influenza A virus. To confirm the immunogenicity of this epitope, genetic manipulating techniques have been used to prepare the recombinant epitope in E. coli as a fusion form with H:1,2 flagellin antigen from Salmonella Typhimurium and with glutathione S-transferase. This recombinant antigen has been collected, purified and used for immunizing mice. Using ELISA method, we could successfully prove that the antirecombinant GST-H:1,2-HeBc antibodies could recognize and bind specifically to the HA antigen derived from an influenza H5N1 virus strain isolated from infected chickens in Vietnam in the same way as did the antiserum from mice immunized with commercial inactived H5N1 influenza vaccine.

Plac1 is a tumor‐specific antigen capable of eliciting spontaneous antibody responses in human cancer patients

Immunoselection and tumor evasion constitutes one of the major obstacles in cancer immunotherapy. A potential solution to this problem is the development of polyvalent vaccines, and the identification of more tumor-specific antigens is a prerequisite for the development of cancer vaccines. To identify novel tumor-specific antigens, suppression subtractive hybridization (SSH) was performed to isolate genes differentially expressed in human hepatocellular cancer (HCC) tissues. PLAC1 (PLACenta-specific 1) was one of the genes identified highly expressed in HCC tissues but not in paired noncancerous tissues. Further analyses revealed its expression in several other types of cancer tissues as well as tumor cell lines, but not in normal tissues except for placenta. Among HCC samples tested, 32% (22/69) showed PLAC1 mRNA expression while the protein was detected in 23.3% (7/30). A serological survey revealed that 3.8% (4/101) of HCC patients had anti-PLAC1 antibody response, suggesting the immunogenicity of PLAC1 in HCC patients. PLAC1 represents a new class of tumor associated antigen with restricted expression in placenta and cancer tissues, that may serve as a target for cancer vaccination.

BORIS, a novel cancer-testis antigen, is a potential target for immunotherapy in epithelial ovarian cancer

9673 Background: Cancer-testis antigens (CTA), are selectively expressed in various types of human neoplasms but not in normal tissues other than testis. This characteristic feature of CTA makes them promising targets for cancer-specific immunotherapy. Brother of Regulator Imprinted Sites (BORIS), a novel CTA located on chromosome 20q13.2, is involved in epigenetic re-programming. The aims of this study were to determine the frequency of aberrant expression of BORIS in epithelial ovarian cancer (EOC) and examine its correlation with clinical outcome. Methods: One step RT-PCR was performed with RNA from various normal and 94 EOC tumor tissues obtained from 1995 to 2002. A 270bp BORIS PCR product was amplified using specific sense 5’-CAGGCCCTACAAGTGTAACGACTGCAA and antisense 5’-GCATTCGTAAGGCTTCTCACCTGAGTG primers. Glyceraldehyde-3-phosphodehydrogenase (GAPDH) specific PCR amplification was used as control The χ2 test was used to analyze the distribution of BORIS expression and clinical outcome. Survival distributions were calculated by the Kaplan-Meier method and statistical significance was determined with the log-rank test. Results: Aberrant expression of BORIS was present in (63/ 94) 67% of EOC primary tumors, and undetectable in a panel of 16 normal tissues. The normal ovarian surface epithelial cell lines, IOSE and HOSE were negative for BORIS while 3/4 (75%) ovarian cancer cell lines (OVCA43, OVCA432, SKOV3) were positive. The median follow up of the patient population was 25 months (range 0.5–119). Tumor expression of BORIS did not correlate with stage, grade or histology. The median survival for BORIS positive patients was 49 months (CI:35,64) compared with 40 months (CI:26,54) for BORIS negative patients (not significant). Conclusion: BORIS is a novel CTA with high frequency expression in EOC. Although the expression of this antigen does not appear to influence survival, the tissue restricted expression pattern, role in epigenetic re-programming, and potential immunogenicity makes BORIS an attractive target for antigen-specific immunotherapy in EOC. The characterization of additional CTA with a frequent expression in EOC is warranted to allow for the development of polyvalent vaccines. No significant financial relationships to disclose.

Book Review: Cancer Vaccines and Immunotherapy

An increasing ability to identify tumour antigens, coupled with advances in immunization, has generated new enthusiasm for cancer vaccination strategies. Cancer Vaccines and Immunotherapy describes the underlying mechanisms and the therapeutic potential of this approach. The short and lucid introduction is essential for an understanding of what follows: it deals with immunosurveillance and how it may be affected in cancer, with immune-mediated elimination of allogeneic tumours in animal models, with the successful treatment of Epstein—Barr virus related lymphoma in renal transplant patients by infusion of immune T cells, and with the way the immune system recognizes and processes tumour antigens, what induces self-tolerance, and which additional molecules are required for an effective immune response. A chapter on pox viruses, one of the principal delivery vehicles for tumour vaccines, serves as useful background to the discussion of clinical trials. The hope, of course, is that vaccination will improve on the results of conventional therapies, especially for common tumours such as breast cancer. In this respect there has been much interest in the MUC1 gene, which encodes for an epithelial mucin overexpressed in malignant breast cells and associated with an altered pattern of glycosylation (though the relevance of this to cell behaviour is unknown). MUC1 can also act as a ligand for the adhesion molecule ICAM-1 and enhances antigen presentation to T cells, possibly via interaction with a lectin molecule. A multitude of antibodies have been raised to MUC1 and some are being used in clinical trials of immunotherapy for breast and ovarian cancer. Early results are promising and phase III studies are underway. One of the key chapters deals with an approach termed SEREX (serological analysis of tumour antigens by recombinant cDNA expression cloning). This involves the construction of cDNA expression libraries from fresh tumour specimens and the selection of clones that elicit high-titre IgG antibody responses. Some of the advantages of this technique are that tumour lines do not have to be established, identified genes are expressed in vivo and multiple antigens can be identified with one screening course and sequenced immediately. By 1998 there were over nine hundred entries on the SEREX database. This technique may allow mapping of the antigenic profile of individual tumours, identification of the antigens that elicit an immune response in cancer patients, and the development of polyvalent vaccines. Although techniques of this kind offer hope that novel treatments will soon emerge, the overview in Cancer Vaccines and Immunotherapy sounds some notes of caution. Of the antigens identified by SEREX few have reached the stage of preclinical trial, so the full limitations of the method have probably yet to emerge. Of the various delivery mechanisms for vaccines, all have advantages and disadvantages. And the remaining challenge is to optimize the design of clinical trials to ensure that vaccines are not only immunogenic and effective but also safe. This requires development of better tools for assessing immunological and clinical aspects. The choice of malignant target and stage of disease is also important. Despite all the scientific advances there is still no certainty that cancer vaccines will work efficiently. The vaccine for which the evidence is strongest, BCG for bladder cancer, is based on the old non-specific approach. But there is sufficient evidence in this valuable survey to warrant a degree of optimism.