Opsonic and Protective Properties of Antibodies Raised to Conjugate Vaccines Targeting Six Staphylococcus aureus Antigens

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections for which a vaccine is greatly desired. Antigens found on the S. aureus outer surface include the capsular polysaccharides (CP) of serotype 5 (CP5) or 8 (CP8) and/or a second antigen, a β-(1→6)-polymer of N-acetyl-D-glucosamine (PNAG). Antibodies specific for either CP or PNAG antigens have excellent in vitro opsonic killing activity (OPKA), but when mixed together have potent interference in OPKA and murine protection. To ascertain if this interference could be abrogated by using a synthetic non-acetylated oligosaccharide fragment of PNAG, 9GlcNH₂, in place of chemically partially deacetylated PNAG, three conjugate vaccines consisting of 9GlcNH₂ conjugated to a non-toxic mutant of alpha-hemolysin (Hla H35L), CP5 conjugated to clumping factor B (ClfB), or CP8 conjugated to iron-surface determinant B (IsdB) were used separately to immunize rabbits. Opsonic antibodies mediating killing of multiple S. aureus strains were elicited for all three vaccines and showed carbohydrate antigen-specific reductions in the tissue bacterial burdens in animal models of S. aureus skin abscesses, pneumonia, and nasal colonization. Carrier-protein specific immunity was also shown to be effective in reducing bacterial levels in infected lungs and in nasal colonization. However, use of synthetic 9GlcNH₂ to induce antibody to PNAG did not overcome the interference in OPKA engendered when these were combined with antibody to either CP5 or CP8. Whereas each individual vaccine showed efficacy, combining antisera to CP antigens and PNAG still abrogated individual OPKA activities, indicating difficulty in achieving a multi-valent vaccine targeting both the CP and PNAG antigens.     

Improved protective efficacy of a chimeric Staphylococcus aureus vaccine candidate iron‐regulated surface determinant B (N126–P361)‐target of RNAIII activating protein in mice

The pathogen Staphylococcus aureus causes a wide range of serious infections, necessitating urgent development of a vaccine against this organism. However, currently developed vaccines are relatively ineffective because of the limited antigenic component that is contained in the vaccine formulations. To develop an effective S. aureus candidate vaccine, overlapping PCR was used to add the truncated immunodominant antigen iron‐regulated surface determinant B (IsdB)_{(N126–P361)} (tIsdB) to the N‐terminal of intact antigen target of RNAIII activating protein (TRAP) and thus construct a tIsdB‐TRAP chimera. The humoral and cellular immune responses against tIsdB‐TRAP were compared with those against single or combined formulations. tIsdB‐TRAP elicited significantly stronger humoral responses in mice (P < 0.05). As to cellular immune responses in mice, the tIsdB‐TRAP group resulted in a greater IL‐4 response than did other groups (P < 0.05). Greater amounts of IL‐2 and IFN‐γ were found in the tIsdB‐TRAP group. Mouse challenge also showed that tIsdB‐TRAP provided better protection against S. aureus than did the control groups. These results suggest that this chimeric protein may be a promising pathogen target for further vaccine development.     

Identification of the Immunodominant Regions of Staphylococcus aureus Fibronectin-Binding Protein A

Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a diverse spectrum of human diseases and a leading cause of nosocomial and community-acquired infections. Development of a vaccine against this pathogen is an important goal. The fibronectin binding protein A (FnBPA) of S. aureus is one of multifunctional ‘microbial surface components recognizing adhesive matrix molecules'' (MSCRAMMs). It is one of the most important adhesin molecules involved in the initial adhesion steps of S. aureus infection. It has been studied as potential vaccine candidates. However, FnBPA is a high-molecular-weight protein of 106 kDa and difficulties in achieving its high-level expression in vitro limit its vaccine application in S. aureus infection diseases control. Therefore, mapping the immunodominant regions of FnBPA is important for developing polyvalent subunit fusion vaccines against S. aureus infections. In the present study, we cloned and expressed the N-terminal and C-terminal of FnBPA. We evaluated the immunogenicity of the two sections of FnBPA and the protective efficacy of the two truncated fragments vaccines in a murine model of systemic S. aureus infection. The results showed recombinant truncated fragment F1_30-500 had a strong immunogenicity property and survival rates significantly increased in the group of mice immunized with F1_30-500 than the control group. We futher identified the immunodominant regions of FnBPA. The mouse antisera reactions suggest that the region covering residues 110 to 263 (F1B_110-263) is highly immunogenic and is the immunodominant regions of FnBPA. Moreover, vaccination with F1B_110-263 can generate partial protection against lethal challenge with two different S. aureus strains and reduced bacterial burdens against non-lethal challenge as well as that immunization with F1_30-500. This information will be important for further developing anti- S. aureus polyvalent subunit fusion vaccines.     

Iron-Regulated Surface Determinant (Isd) Proteins of Staphylococcus lugdunensis

Staphylococcus lugdunensis is the only coagulase-negative Staphylococcus species with a locus encoding iron-regulated surface determinant (Isd) proteins. In Staphylococcus aureus, the Isd proteins capture heme from hemoglobin and transfer it across the wall to a membrane-bound transporter, which delivers it into the cytoplasm, where heme oxygenases release iron. The Isd proteins of S. lugdunensis are expressed under iron-restricted conditions. We propose that S. lugdunensis IsdB and IsdC proteins perform the same functions as those of S. aureus. S. lugdunensis IsdB is the only hemoglobin receptor within the isd locus. It specifically binds human hemoglobin with a dissociation constant (K_d) of 23 nM and transfers heme on IsdC. IsdB expression promotes bacterial growth in an iron-limited medium containing human hemoglobin but not mouse hemoglobin. This correlates with weak binding of IsdB to mouse hemoglobin in vitro. Unlike IsdB and IsdC, the proteins IsdJ and IsdK are not sorted to the cell wall in S. lugdunensis. In contrast, IsdJ expressed in S. aureus and Lactococcus lactis is anchored to peptidoglycan, suggesting that S. lugdunensis sortases may differ in signal recognition or could be defective. IsdJ and IsdK are present in the culture supernatant, suggesting that they could acquire heme from the external milieu. The IsdA protein of S. aureus protects bacteria from bactericidal lipids due to its hydrophilic C-terminal domain. IsdJ has a similar region and protected S. aureus and L. lactis as efficiently as IsdA but, possibly due to its location, was less effective in its natural host.     

Evaluation of Genetically Inactivated Alpha Toxin for Protection in Multiple Mouse Models of Staphylococcus aureus Infection

Staphylococcus aureus is a major human pathogen and a leading cause of nosocomial and community-acquired infections. Development of a vaccine against this pathogen is an important goal. While S. aureus protective antigens have been identified in the literature, the majority have only been tested in a single animal model of disease. We wished to evaluate the ability of one S. aureus vaccine antigen to protect in multiple mouse models, thus assessing whether protection in one model translates to protection in other models encompassing the full breadth of infections the pathogen can cause. We chose to focus on genetically inactivated alpha toxin mutant HlaH35L. We evaluated the protection afforded by this antigen in three models of infection using the same vaccine dose, regimen, route of immunization, adjuvant, and challenge strain. When mice were immunized with HlaH35L and challenged via a skin and soft tissue infection model, HlaH35L immunization led to a less severe infection and decreased S. aureus levels at the challenge site when compared to controls. Challenge of HlaH35L-immunized mice using a systemic infection model resulted in a limited, but statistically significant decrease in bacterial colonization as compared to that observed with control mice. In contrast, in a prosthetic implant model of chronic biofilm infection, there was no significant difference in bacterial levels when compared to controls. These results demonstrate that vaccines may confer protection against one form of S. aureus disease without conferring protection against other disease presentations and thus underscore a significant challenge in S. aureus vaccine development.     

Enhanced Interaction of Shuffled Mutacin IV,an Antimicrobial Peptide of Bacterial Origin,with Surface Protein IsdB of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Antimicrobial peptides are produced by prokaryotes and eukaryotes with fundamental role of protection against pathogenic microbes. Staphylococcus aureus, a major virulent pathogen in humans, shows multiple drug resistance and is affected by the bacteriocin activity of Mutacin IV. Currently, peptide therapeutics has been reported as a potential alternative for treating microbial infections specially exhibiting multiple drug resistance. However, the mechanism of action and interaction of peptides with target proteins is not known. The current work is an attempt to address the above issue by performing molecular docking and randomization experiments. In this study, antimicrobial peptides of bacterial origin (168 peptides) were collected from APD2 database and their net charge and hydrophobicity values were retrieved. Mutacin IV (APD Id—AP01174), a 44 amino acids long peptide derived from Streptococcus mutans UA140, was selected on the basis of high hydrophobicity to net charge ratio (0.52) and used for in silico docking studies with therapeutically important surface proteins viz. IsdA, IsdB, ClfB, and SasG of S. aureus using ZDOCK server. The docking result of IsdB surface protein and Mutacin IV was found better (ZDOCK score 1168.582) as compared to others. Afterwards, the native Mutacin IV sequence was randomized to generate 50 new combinations using EMBOSS (Shuffleseq) tool. The new sequence of Mutacin IV was screened on the basis of high in vivo to in vitro aggregation ratio (i.e. high in vivo aggregation and low in vitro aggregation values) and good binding energies against IsdB surface protein of S. aureus from the randomized sequences. The new peptide sequence showed an in vivo to in vitro aggregation ratio of 2.206 and 0.888, respectively which is higher than native sequence of Mutacin IV ratio (0.205). Moreover, the ZDOCK scores were found to be 1370.529 and 1687.048 which were better than the native sequence of Mutacin IV (ZDOCK score 1168.582). This research work identifies the new sequence of Mutacin IV peptide which binds effectively to the surface proteins of S. aureus and thereby could be a better peptide than native Mutacin IV. Our finding also demonstrates enhanced interactions of new Mutacin IV peptide with IsdB surface protein to understand the structural implications and proposes its effective antimicrobial role against S. aureus.     

Reverse-vaccinology strategy for designing T-cell epitope candidates for Staphylococcus aureus endocarditis vaccine

Staphylococcus aureus is an opportunistic pathogen causing various inflammatory diseases from skin and tissue local infections, to serious life threatening infections including endocarditis. Experimental models for endocarditis demonstrated that virulence factors of S. aureus, that are very important in infection of heart vegetations, are surface proteins which promote bacterial adherence. Until now, efforts to develop effective vaccines against S. aureus were unsuccessful, partly due to the fact that different vaccine formulations have targeted mainly B-cell immunity. Reverse vaccinology is applied here, in order to identify potential vaccine epitope candidates. The basic epitopes prediction strategy relied on detection of a common antigenic 9-mer epitope meant to be able to stimulate both the B-cell and T-cell mediated immunity. Ten surface exposed proteins were chosen for antigenicity testing. Using a web-based system, five T-cell epitopes corresponding to fibronectin binding protein A (FDFTLSNNV and YVDGYIETI), collagen adhesin (FSINYKTKI), serine-rich adhesin for platelets (LTFDSTNNT) and elastin binding protein (FAMDKSHPE) were selected as potential vaccine candidates. Epitopes sequences were found to be conserved among the different S. aureus genomes screened from NCBI GenBank. In vitro and in vivo immunological tests will be performed in order to validate the suitability of the epitopes for vaccine development.     

1H, 13C, 15N backbone and side chain NMR resonance assignments of the N-terminal NEAr iron transporter domain 1 (NEAT 1) of the hemoglobin receptor IsdB of Staphylococcus aureus

Staphylococcus aureus is an opportunistic pathogen that causes skin and severe infections in mammals. Critical to S. aureus growth is its ability to scavenge iron from host cells. To this effect, S. aureus has evolved a sophisticated pathway to acquire heme from hemoglobin (Hb) as a preferred iron source. The pathway is comprised of nine iron-regulated surface determinant (Isd) proteins involved in heme capture, transport, and degradation. A key protein of the heme acquisition pathway is the surface-anchored hemoglobin receptor protein IsdB, which is comprised of two NEAr transporter (NEAT) domains that act in concert to bind Hb and extract heme for subsequent transfer to downstream acquisition pathway proteins. Despite significant advances in the structural knowledge of other Isd proteins, the structural mechanisms and molecular basis of the IsdB-mediated heme acquisition process are not well understood. In order to provide more insights into the mode of function of IsdB, we have initiated NMR structural studies of the first NEAT domain of IsdB (IsdB^N1). Herein, we report the near complete ¹H, ¹³C and ¹⁵N resonance assignments of backbone and side chain atoms, and the secondary structural topology of the 148-residue IsdB NEAT 1 domain. The NMR results are consistent with the presence of eight β-strands and one α-helix characteristic of an immunoglobulin-like fold observed in other NEAT domain family proteins. This work provides a solid framework to obtain atomic-level insights toward understanding how IsdB mediates IsdB-Hb protein–protein interactions critical for heme capture and transfer.     

Bivalent Vaccines Against Bacterial Enteropathogens: Construction of Live Attenuated Vaccine Strains With Two O-Serotype Specificities

Abstract. A considerable interest exists worldwide in the development of live attenuated oral vaccines against diarrhoeal diseases. In addition to vaccination against the corresponding pathogens, such vaccine strains can be used as carriers for the expression of protective antigens from other organisms. The antigenic repertoire of a given vaccine strain may thereby be extended, potentially leading to a bivalent vaccine. The lipopolysaccharide is known to be a major antigenic surface component of bacterial enteric pathogens. The feasibility of the development of combined vaccines based on live attenuated carriers expressing two O-serotype specificities is illustrated here by the development of candidate live oral vaccines against Shigella sonnei using Salmonella typhi and Vibrio cholerae as carriers. Various factors that may limit the potential of such hybrid strains as bivalent vaccines are discussed.     

Newcastle Disease Virus Fusion Protein Is the Major Contributor to Protective Immunity of Genotype-Matched Vaccine

Virulent strains of Newcastle disease virus (NDV) can cause devastating disease in chickens worldwide. Although the current vaccines are substantially effective, they do not completely prevent infection, virus shedding and disease. To produce genotype-matched vaccines, a full-genome reverse genetics system has been used to generate a recombinant virus in which the F protein cleavage site has been changed to that of avirulent vaccine virus. In the other strategy, the vaccines have been generated by replacing the F and HN genes of a commercial vaccine strain with those from a genotype-matched virus. However, the protective efficacy of a chimeric virus vaccine has not been directly compared with that of a full-genome virus vaccine developed by reverse genetics. Therefore, in this study, we evaluated the protective efficacy of genotype VII matched chimeric vaccines by generating three recombinant viruses based on avirulent LaSota (genotype II) strain in which the open reading frames (ORFs) encoding the F and HN proteins were replaced, individually or together, with those of the circulating and highly virulent Indonesian NDV strain Ban/010. The cleavage site of the Ban/010 F protein was mutated to the avirulent motif found in strain LaSota. In vitro growth characteristics and a pathogenicity test indicated that all three chimeric viruses retained the highly attenuated phenotype of the parental viruses. Immunization of chickens with chimeric and full-length genome VII vaccines followed by challenge with virulent Ban/010 or Texas GB (genotype II) virus demonstrated protection against clinical disease and death. However, only those chickens immunized with chimeric rLaSota expressing the F or F plus HN proteins of the Indonesian strain were efficiently protected against shedding of Ban/010 virus. Our findings showed that genotype-matched vaccines can provide protection to chickens by efficiently preventing spread of virus, primarily due to the F protein.     

PAMAM-Lys,a Novel Vaccine Delivery Vector,Enhances the Protective Effects of the SjC23 DNA Vaccine against Schistosoma japonicum Infection

Background

Schistosomiasis japonica remains a major public-health concern in China. Praziquantel-based chemotherapy effectively reduces both infections and intensity; however, it can not prevent re-infection. Furthermore, there is an increasing concern about praziquantel resistance following long-term repeated use of the drug in endemic areas. Therefore, development of a schistosomiasis vaccine, as a strategy to prevent and control schistosomiasis japonica, has been given high priority. The present study was conducted to develop PAMAM dendrimers as a novel vaccine delivery vector for a schistosomiasis japonica DNA vaccine and evaluate its ability to enhance protective effects against Schistosoma japonicum infection.

Methodology/Principal Findings

Lysine was used to modify 4.0G PAMAM, and the modified product PAMAM-Lys was synthesized. PAMAM-Lys showed both high transfection and low cytotocity for gene delivery in vitro. DNA vaccines combined with PAMAM-Lys produced higher level of protection compare with naked DNA vaccines against S. japonicum infection in a mouse model. Futhermore,antibodies from mice immunized with PAMAM-Lys combined DNA vaccines were significantly higher than those of mice immunized with the naked DNA vaccines. The PAMAM-Lys vector elicited a predominantly IgG2a antibody response and a tremendously increase in the production of IL-2 and IFN-γ.

Conclusion/Significance

Lysine-modified PAMAM-Lys is an excellent vector. PAMAM-Lys may enhance the immunoreactivity of DNA vaccine and increase the protective effect of the SjC23 DNA vaccine against S. japonicum infection.     

cAMP and EPAC Are Key Players in the Regulation of the Signal Transduction Pathway Involved in the α-Hemolysin Autophagic Response

Staphylococcus aureus is a microorganism that causes serious diseases in the human being. This microorganism is able to escape the phagolysosomal pathway, increasing intracellular bacterial survival and killing the eukaryotic host cell to spread the infection. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. We have shown that the pore-forming toxin α-hemolysin (Hla) is the S. aureus–secreted factor responsible for the activation of the autophagic pathway and that this response occurs through a PI3K/Beclin1-independent form. In the present report we demonstrate that cAMP has a key role in the regulation of this autophagic response. Our results indicate that cAMP is able to inhibit the autophagy induced by Hla and that PKA, the classical cAMP effector, does not participate in this regulation. We present evidence that EPAC and Rap2b, through calpain activation, are the proteins involved in the regulation of Hla-induced autophagy. Similar results were obtained in cells infected with different S. aureus strains. Interestingly, in this report we show, for the first time to our knowledge, that both EPAC and Rap2b are recruited to the S. aureus–containing phagosome. We believe that our findings have important implications in understanding innate immune processes involved in intracellular pathogen invasion of the host cell.     

Identification and Validation of a Linear Protective Neutralizing Epitope in the β-Pore Domain of Alpha Toxin

The plethora of virulence factors associated with Staphylococcus aureus make this bacterium an attractive candidate for a molecularly-designed epitope-focused vaccine. This approach, which necessitates the identification of neutralizing epitopes for incorporation into a vaccine construct, is being evaluated for pathogens where conventional approaches have failed to elicit protective humoral responses, like HIV-1 and malaria, but may also hold promise for pathogens like S. aureus, where the elicitation of humoral immunity against multiple virulence factors may be required for development of an effective vaccine. Among the virulence factors employed by S. aureus, animal model and epidemiological data suggest that alpha toxin, a multimeric β-pore forming toxin like protective antigen from Bacillus anthracis, is particularly critical, yet no candidate neutralizing epitopes have been delineated in alpha toxin to date. We have previously shown that a linear determinant in the 2β2-2β3 loop of the pore forming domain of B. anthracis protective antigen is a linear neutralizing epitope. Antibody against this site is highly potent for neutralizing anthrax lethal toxin in vitro and for protection of rabbits in vivo from virulent B. anthracis. We hypothesized that sequences in the β-pore of S. aureus alpha toxin that share structural and functional homology to β-pore sequences in protective antigen would contain a similarly critical neutralizing epitope. Using an in vivo mapping strategy employing peptide immunogens, an optimized in vitro toxin neutralization assay, and an in vivo dermonecrosis model, we have now confirmed the presence of this epitope in alpha toxin, termed the pore neutralizing determinant. Antibody specific for this determinant neutralizes alpha toxin in vitro, and is highly effective for mitigating dermonecrosis and bacterial growth in a mouse model of S. aureus USA300 skin infection. The delineation of this linear neutralizing determinant in alpha toxin could facilitate the development of an epitope-focused vaccine against S. aureus.     

Staphylococcus aureus promotes autophagy by decreasing intracellular cAMP levels

Staphylococcus aureus is an intracellular bacterium responsible for serious infectious processes. This pathogen escapes from the phagolysosomal pathway into the cytoplasm, a strategy that allows intracellular bacterial replication and survival with the consequent killing of the eukaryotic host cell and spreading of the infection. S. aureus is able to secrete several virulence factors such as enzymes and toxins. Our recent findings indicate that the main virulence factor of S. aureus, the pore-forming toxin α-hemolysin (Hla), is the secreted factor responsible for the activation of an alternative autophagic pathway. We have demonstrated that this noncanonical autophagic response is inhibited by artificially elevating the intracellular levels of cAMP. This effect is mediated by RAPGEF3/EPAC (Rap guanine nucleotide exchange factor (GEF)3/exchange protein activated by cAMP), a cAMP downstream effector that functions as a GEF for the small GTPase Rap. We have presented evidence that RAPGEF3 and RAP2B, through calpain activation, are the proteins involved in the regulation of Hla and S. aureus-induced autophagy. In addition, we have found that both, RAPGEF3 and RAP2B, are recruited to the S. aureus–containing phagosome. Of note, adding purified α-toxin or infecting the cells with S. aureus leads to a decrease in intracellular cAMP levels, which promotes autophagy induction, a response that favors pathogen intracellular survival, as previously demonstrated. We have identified some key signaling molecules involved in the autophagic response upon infection with a bacterial pathogen, which have important implications in understanding innate immune defense mechanisms.     

Novel structurally designed vaccine for S. aureus α-hemolysin: protection against bacteremia and pneumonia

Staphylococcus aureus (S. aureus) is a human pathogen associated with skin and soft tissue infections (SSTI) and life threatening sepsis and pneumonia. Efforts to develop effective vaccines against S. aureus have been largely unsuccessful, in part due to the variety of virulence factors produced by this organism. S. aureus alpha-hemolysin (Hla) is a pore-forming toxin expressed by most S. aureus strains and reported to play a key role in the pathogenesis of SSTI and pneumonia. Here we report a novel recombinant subunit vaccine candidate for Hla, rationally designed based on the heptameric crystal structure. This vaccine candidate, denoted AT-62aa, was tested in pneumonia and bacteremia infection models using S. aureus strain Newman and the pandemic strain USA300 (LAC). Significant protection from lethal bacteremia/sepsis and pneumonia was observed upon vaccination with AT-62aa along with a Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE) that is currently in clinical trials. Passive transfer of rabbit immunoglobulin against AT-62aa (AT62-IgG) protected mice against intraperitoneal and intranasal challenge with USA300 and produced significant reduction in bacterial burden in blood, spleen, kidney, and lungs. Our Hla-based vaccine is the first to be reported to reduce bacterial dissemination and to provide protection in a sepsis model of S. aureus infection. AT62-IgG and sera from vaccinated mice effectively neutralized the toxin in vitro and AT62-IgG inhibited the formation of Hla heptamers, suggesting antibody-mediated neutralization as the primary mechanism of action. This remarkable efficacy makes this Hla-based vaccine a prime candidate for inclusion in future multivalent S. aureus vaccine. Furthermore, identification of protective epitopes within AT-62aa could lead to novel immunotherapy for S. aureus infection.     

Identification of novel vaccine candidate against Salmonella enterica serovar Typhi by reverse vaccinology method and evaluation of its immunization

Salmonella enterica serovar Typhi (S. Typhi) is an essential enteric fever causing bacterium worldwide. Due to the emergence of multidrug-resistant strains, urgently attention is needed to prevent the global spread of them. Vaccination is an alternative approach to control these kinds of infections. Currently available commercial vaccines have significant limitations such as non-recommendation for children below six years of age and poor long-term efficacy. Thus, the development of a new vaccine overcoming these limitations is immediately required. Reverse Vaccinology (RV) is one of the most robust approaches for direct screening of genome sequence assemblies to identify new protein-based vaccines. The present study aimed to identify potential vaccine candidates against S. Typhi by using the RV approach. Immunogenicity of the best candidate against S. Typhi was further investigated. The proteome of S. Typhi strain Ty2 was analyzed to identify the most immunogenic, conserved, and protective surface proteins. Among the predicted vaccine candidates, steD (fimbrial subunit) was the best for qualifying all the applied criteria. The synthetic steD gene was expressed in E.coli, and the mice were immunized with purified recombinant steD protein and then challenged with a lethal dose of S. Typhi. Immunized animals generated high protein-specific antibody titers and demonstrated 70% survival following lethal dose challenge with S. Typhi. Pretreatment of the S. Typhi cells with immunized mice antisera significantly decreased their adhesion to Caco-2 cells. Altogether, steD as a protective antigen could induce a robust and long term and protective immunity in immunized mice against S. Typhi challenge.     

Chimeric animal and plant viruses expressing epitopes of outer membrane protein F as a combined vaccine against Pseudomonas aeruginosa lung infection

Outer membrane protein F of Pseudomonas aeruginosa has vaccine efficacy against infection by P. aeruginosa as demonstrated in a variety of animal models. Through the use of synthetic peptides, three surface-exposed epitopes have been identified. These are called peptides 9 (aa 261-274 in the mature F protein, TDAYNQKLSERRAN), 10 (aa 305-318, NATAEGRAINRRVE), and 18 (aa 282-295, NEYGVEGGRVNAVG). Both the peptide 9 and 10 epitopes are protective when administered as a vaccine. In order to develop a vaccine that is suitable for use in humans, including infants with cystic fibrosis, the use of viral vector systems to present the protective epitopes has been investigated. An 11-amino acid portion of epitope 10 (AEGRAINRRVE) was successfully inserted into the antigenic B site of the hemagglutinin on the surface of influenza virus. This chimeric influenza virus protects against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Attempts to derive a chimeric influenza virus carrying epitope 9 have been unsuccessful. A chimeric plant virus, cowpea mosaic virus (CPMV), with epitopes 18 and 10 expressed in tandem on the large coat protein subunit (CPMV-PAE5) was found to elicit antibodies that reacted exclusively with the 10 epitope and not with epitope 18. Use of this chimeric virus as a vaccine afforded protection against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Chimeric CPMVs with a single peptide containing epitopes 9 and 18 expressed on either of the coat proteins are in the process of being evaluated. Epitope 9 was successfully expressed on the coat protein of tobacco mosaic virus (TMV), and this chimeric virus is protective when used as a vaccine in the mouse model of chronic pulmonary infection. However, initial attempts to express epitope 10 on the coat protein of TMV have been unsuccessful. Efforts are continuing to construct chimeric viruses that express both the 9 and 10 epitopes in the same virus vector system. Ideally, the use of a vaccine containing two epitopes of protein F is desirable in order to greatly reduce the likelihood of selecting a variant of P. aeruginosa that escapes protective antibodies in immunized humans via a mutation in a single epitope within protein F. When the chimeric influenza virus containing epitope 10 and the chimeric TMV containing epitope 9 were given together as a combined vaccine, the immunized mice produced antibodies directed toward both epitopes 9 and 10. The combined vaccine afforded protection against challenge with P. aeruginosa in the chronic pulmonary infection model at approximately the same level of efficacy as provided by the individual chimeric virus vaccines. These results prove in principle that a combined chimeric viral vaccine presenting both epitopes 9 and 10 of protein F has vaccine potential warranting continued development into a vaccine for use in humans.     

An integrated in silico based subtractive genomics and reverse vaccinology approach for the identification of novel vaccine candidate and chimeric vaccine against XDR Salmonella typhi H58

Salmonella typhi is notorious for causing enteric fever which is also known as typhoid fever. It emerged as an extreme drug resistant strain that requires urgent attention to prevent its global spread. Statistically, about 11–17 million typhoid illnesses are reported worldwide annually. The only alternative approach for the control of this illness is proper vaccination. However, available typhoid vaccine has certain limitations such as poor long-term efficacy, and non-recommendation for below 6 years children, which opens the avenues for designing new vaccines to overcome such limitations. Computational-based reverse vaccinology along with subtractive genomics analysis is one of the robust approaches used for the prioritization of vaccine candidates through direct screening of genome sequence assemblies. In the current study, we have successfully designed a peptide-based novel antigen chimeric vaccine candidate against the XDR strain of S. typhi H58. The pipeline revealed four peptides from WP_001176621.1 i.e., peptidoglycan-associated lipoprotein Pal and two peptides from WP_000747548.1 i.e., OmpA family lipoprotein as promising target for the induction of immune response against S. typhi. The six epitopes from both proteins were found as immunogenic, antigenic, virulent, highly conserved, nontoxic, and non-allergenic among whole Salmonella H58 proteome. Furthermore, the binding interaction between a chimeric vaccine and human population alleles was unveiled through structure-based studies. So far, these proteins have never been characterized as vaccine targets against S. typhi. The current study proposed that construct V2 could be a significant vaccine candidate against S. typhi H58. However, to ascertain this, future experimental holistic studies are recommended as follow-up.