Cross-protective Vaccine Candidate Research Articles

Shigella virulence protein VirG is a broadly protective antigen and vaccine candidate

Diarrhea caused by Shigella has been associated with high morbidity and mortality in young children worldwide. There are no licensed vaccines, and those clinically advanced have restricted coverage as they elicit serotype-specific immunity while disease is caused by multiple circulating serotypes. Our group had previously reported a close association between serum antibodies to the Shigella virulence factor VirG (or IcsA) and clinical protection in infected individuals. VirG is highly conserved among Shigella strains and appealing as a broad-spectrum vaccine candidate. In this study, we investigated the immunogenicity and protective capacity of VirG as a subunit vaccine in mice. The surface-exposed alpha (α) domain of VirG (VirGα) was produced as a recombinant protein. This region has almost identical immune reactivity to full-length VirG. Administered intramuscularly with alum, VirGα elicited robust immune responses and high protective efficacy against S. flexneri 2a and S. sonnei. Almost complete protection was afforded by VirGα given intranasally with the E. coli double mutant heat-labile toxin (dmLT). VirGα-specific antibodies recognized VirG expressed on live Shigella, and blocked Shigella adhesion and invasion to human colonic cells. These results show for the first time that VirGα is a promising cross-protective vaccine candidate to prevent Shigella infection.

Designing and Computational Analysis of Chimeric Avian Influenza Antigen: A Yeast-Displayed Universal and Cross-Protective Vaccine Candidate

This study describes the development of a cross-protective vaccine candidate against avian influenza virus, which was designed using M2e, a highly preserved antigen. The consensus sequence of M2e was obtained using 31 sequences of avian influenza virus subtypes (H5N8, H5N1, H9N2, and H7N9) isolated from seven avian species in five Asian countries. An adjuvant, a partial sequence of flagellin, was also considered. Two chimeric antigens were designed and virtually cloned and expressed using the PYD1 vector and EBY100 yeast strain. Molecular dynamic simulations were used to assess the stability and conformational features of these antigens. The likelihood of detection by a specific monoclonal antibody, MAb148, was estimated for the designed peptides using docking studies. The second chimeric antigen was more compact and stable than the first design, but it was less detectable by MAb148. In the first design, two of the four desired epitopes ("SLLTEVETP") were exposed, while only a partial sequence of this epitope was detectable in the second design. In contrast to the second chimeric antigen, electrostatic, and binding energies related to the interaction of the first antigen and MAb148 were significantly closer to the positive control. This suggests that epitopes of the first chimeric antigen could be correctly located in the specific paratope of MAb148. In conclusion, the first chimeric antigen exhibits favorable conformational features and epitope-paratope interactions, highlighting its potential as a promising cross-protective vaccine candidate against a range of avian influenza virus subtypes.

Development of a Self-Adjuvanting, Cross-Protective, Stable Intranasal Recombinant Vaccine for Shigellosis.

With the acquirement of antibiotic resistance, Shigella has resulted in multiple epidemics of shigellosis, an infectious diarrheal disease, causing thousands of deaths per year. Unfortunately, there are no licensed vaccines, primarily due to low or serotype-specific immunogenicity. Thus, conserved subunit vaccines utilizing recombinant invasion plasmid antigens (Ipa) have been explored as cross-protective vaccine candidates. However, achieving cross-protection against Shigella dysenteriae 1, which caused multiple pandemics/epidemics in the recent past, has been difficult. Therefore, a rational approach to improve cross-protection in the preparation for a possible pandemic should involve conserved proteins from S. dysenteriae 1 (Sd1). IpaC is one such conserved immunogenic protein that is less explored as an independent vaccine due to its instability/aggregation. Therefore, to improve cross-protection and potential immunogenicity and to be prepared for a future epidemic/pandemic, herein, we stabilized recombinant Sd1 IpaC, expressed without its chaperone, using a previously reported stabilizing detergent (LDAO) in a modified protocol and assessed its vaccine potential without an adjuvant. The protein assembled into heterogeneous complex spherical structures in the presence of LDAO and showed improved stability at storage temperatures of -80, -20, 4, 25, and 37 °C while providing enhanced yield and concentration. The protein could also be stably lyophilized and reconstituted, increasing the convenience of transportation and storage. Upon intranasal administration in BALB/c mice, the stabilized-IpaC-immunized groups generated significant antibody response and were not only protected against a high intraperitoneal dose of homologous S. dysenteriae 1 but also showed 100% survival against heterologous Shigella flexneri 2a without an adjuvant, while the control animals showed visible diarrhea (bloody-Sd1 challenge), lethargy, and weight loss with 0% survival. Overall, this work demonstrates that stabilized IpaC can be explored as a minimalist, self-adjuvanting, cross-protective, intranasal, single-antigen Shigella vaccine.

Cowpea Mosaic Virus Nanoparticle Vaccine Candidates Displaying Peptide Epitopes Can Neutralize the Severe Acute Respiratory Syndrome Coronavirus.

The development of vaccines against coronaviruses has focused on the spike (S) protein, which is required for the recognition of host-cell receptors and thus elicits neutralizing antibodies. Targeting conserved epitopes on the S protein offers the potential for pan-beta-coronavirus vaccines that could prevent future pandemics. We displayed five B-cell epitopes, originally identified in the convalescent sera from recovered severe acute respiratory syndrome (SARS) patients, on the surface of the cowpea mosaic virus (CPMV) and evaluated these formulations as vaccines. Prime-boost immunization of mice with three of these candidate vaccines, CPMV-988, CPMV-1173, and CPMV-1209, elicited high antibody titers that neutralized the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro and showed an early Th1-biased profile (2–4 weeks) transitioning to a slightly Th2-biased profile just after the second boost (6 weeks). A pentavalent slow-release implant comprising all five peptides displayed on the CPMV elicited anti-S protein and epitope-specific antibody titers, albeit at a lower magnitude compared to the soluble formulations. While the CPMV remained intact when released from the PLGA implants, processing results in loss of RNA, which acts as an adjuvant. Loss of RNA may be a reason for the lower efficacy of the implants. Finally, although the three epitopes (988, 1173, and 1209) that were found to be neutralizing the SARS-CoV were 100% identical to the SARS-CoV-2, none of the vaccine candidates neutralized the SARS-CoV-2 in vitro suggesting differences in the natural epitope perhaps caused by conformational changes or the presence of N-linked glycans. While a cross-protective vaccine candidate was not developed, a multivalent SARS vaccine was developed. The technology discussed here is a versatile vaccination platform that can be pivoted toward other diseases and applications that are not limited to infectious diseases.

Cross-serotype protection against group A Streptococcal infections induced by immunization with SPy_2191

Group A Streptococcus (GAS) infection causes a range of diseases, but vaccine development is hampered by the high number of serotypes. Here, using reverse vaccinology the authors identify SPy_2191 as a cross-protective vaccine candidate. From 18 initially identified surface proteins, only SPy_2191 is conserved, surface-exposed and inhibits both GAS adhesion and invasion. SPy_2191 immunization in mice generates bactericidal antibodies resulting in opsonophagocytic killing of prevalent and invasive GAS serotypes of different geographical regions, including M1 and M49 (India), M3.1 (Israel), M1 (UK) and M1 (USA). Resident splenocytes show higher interferon-γ and tumor necrosis factor-α secretion upon antigen re-stimulation, suggesting activation of cell-mediated immunity. SPy_2191 immunization significantly reduces streptococcal load in the organs and confers ~76-92% protection upon challenge with invasive GAS serotypes. Further, it significantly suppresses GAS pharyngeal colonization in mice mucosal infection model. Our findings suggest that SPy_2191 can act as a universal vaccine candidate against GAS infections.

Exploiting bacterial outer membrane vesicles as a cross-protective vaccine candidate against avian pathogenic Escherichia coli (APEC)

BackgroundThe well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its numerous serogroups has promoted the development of biological immunostimulatory materials as new vaccine candidates in poultry farms. Bacterial outer membrane vesicles (OMVs), known as spherical nanovesicles enriched with various immunostimulants, are naturally secreted by Gram-negative bacteria, and have gained much attention for developing effective vaccine candidates. Recent report has demonstrated that OMVs of APEC O78 can induce protective immunity in chickens. Here, a novel multi-serogroup OMVs (MOMVs) vaccine was developed to achieve cross-protection against APEC infection in broiler chickens.ResultsIn this study, OMVs produced by three APEC strains were isolated, purified and prepared into MOMVs by mixing these three OMVs. By using SDS-PAGE and LC–MS/MS, 159 proteins were identified in MOMVs and the subcellular location and biological functions of 20 most abundant proteins were analyzed. The immunogenicity of MOMVs was evaluated, and the results showed that MOMVs could elicit innate immune responses, including internalization by chicken macrophage and production of immunomodulatory cytokines. Vaccination with MOMVs induced specific broad-spectrum antibodies as well as Th1 and Th17 immune responses. The animal experiment has confirmed that immunization with an appropriate dose of MOMVs could not cause any adverse effect and was able to reduce bacteria loads and pro-inflammatory cytokines production, thus providing effective cross-protection against lethal infections induced by multi-serogroup APEC strains in chickens. Further experiments indicated that, although vesicular proteins were able to induce stronger protective efficiency than lipopolysaccharide, both vesicular proteins and lipopolysaccharide are crucial in MOMVs-mediated protection.ConclusionsThe multi-serogroup nanovesicles produced by APEC strains will open up a new way for the development of next generation vaccines with low toxicity and broad protection in the treatment and control of APEC infection.

Vaccination with Consensus H7 Elicits Broadly Reactive and Protective Antibodies against Eurasian and North American Lineage H7 Viruses.

H7 subtype avian influenza viruses have caused outbreaks in poultry, and even human infection, for decades in both Eurasia and North America. Although effective vaccines offer the best protection against avian influenza viruses, antigenically distinct Eurasian and North American lineage subtype H7 viruses require the development of cross-protective vaccine candidates. In this study, a methodology called computationally optimized broadly reactive antigen (COBRA) was used to develop four consensus H7 antigens (CH7-22, CH7-24, CH7-26, and CH7-28). In vitro experiments confirmed the binding of monoclonal antibodies to the head and stem domains of cell surface-expressed consensus HAs, indicating display of their antigenicity. Immunization with DNA vaccines encoding the four antigens was evaluated in a mouse model. Broadly reactive antibodies against H7 viruses from Eurasian and North American lineages were elicited and detected by binding, inhibition, and neutralizing analyses. Further infection with Eurasian H7N9 and North American H7N3 virus strains confirmed that CH7-22 and CH7-24 conferred the most effective protection against hetero-lethal challenge. Our data showed that the consensus H7 vaccines elicit a broadly reactive, protective response against Eurasian and North American lineage H7 viruses, which are suitable for development against other zoonotic influenza viruses.

Neuraminidase expressing virus-like particle vaccine provides effective cross protection against influenza virus

Neuraminidase is the second major surface antigen on influenza virus. We investigated the immunogenicity and cross protective efficacy of virus-like particle containing neuraminidase derived from 2009 pandemic H1N1 influenza virus (N1 VLP) in comparison with inactivated split influenza vaccine. Immunization of mice with N1 VLP induced antibody responses specific for virus and cross-reactive neuraminidase inhibition activity whereas an inactivated split vaccine induced strain-specific hemagglutination inhibition activity. N1 VLP-immunized mice developed cross protective immunity against antigenically different influenza viruses, as determined by body weight changes, lung viral titers, infiltrating innate immune cells, and cytokines, and antibody secreting cells, and germinal center B cells. Also, N1 VLP-immune sera provided cross-protection in naïve mice. Immunity by N1 VLP vaccination was not compromised in Fc receptor γ-chain deficient mice. These results suggest that neuraminidase-presenting VLP can be developed as an effective cross-protective vaccine candidate along with current influenza vaccination.

Neuraminidase expressed on virus-like particles is superior to inactivated split virus vaccine in conferring cross protection via humoral and cellular immunity

Abstract Neuraminidase (NA) is the second major surface antigen on influenza virus but often ignored as a vaccine component. In this study, we developed N1 virus-like particles (N1 VLP) containing NA derived from 2009 pandemic H1N1 influenza virus (2009H1N1), and compared with inactivated split virus vaccine in the immune mechanisms of cross protection. Intramuscular immunization of mice with N1 VLP induced IgG antibody responses to 2009H1N1 virus and NA inhibition activity. Mice immunized with N1 VLP induced superior cross protection against lethal infection compared to a current vaccine platform, split virus vaccine (1934H1N1), as determined by body weight changes, lung viral titers, infiltrating innate immune cells, cytokines, and histopathology. Furthermore, N1 VLP immunized mice induced higher levels of IFN-γ effector CD4 T cells, plasma cells, and germinal center B cell (GL7+B220+IgD−CD19+) and plasma cells (CD138+B220+/− IgD-CD19+) than split virus vaccine mice. Also, N1 VLP immune sera provided heterosubtypic cross-protection against H3N2 virus in naïve mice. Therefore, these results suggest that N1 NA-presenting VLP can be developed as an effective cross-protective vaccine candidate standalone or in combination of current influenza vaccination in a supplemented vaccine platform.

The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis

In this study, the Riemerella anatipestifer mutant strain RA1062 was obtained by screening a random Tn4351 transposon mutant library. The mutant strain was unreactive with the anti-CH3 lipopolysaccharide monoclonal antibody, as demonstrated with an enzyme-linked immunosorbent assay, and its M949_RS01035 gene was inactivated. When cultured in trypticase soy broth, the late stage growth of the mutant RA1062 was significantly decreased. The mutant RA1062 was stained with crystal violet and presented a rough lipopolysaccharide phenotype, which differed from that of the wild-type strain CH3, suggesting that deletion of the M949_RS01035 gene resulted in defective lipopolysaccharide. Silver staining and Western blot analyses further confirmed that the RA1062 lipopolysaccharide had a deficiency in ladder-like binding pattern, as compared to lipopolysaccharide of the wild-type CH3 strain. In addition, the mutant RA1062 showed a higher susceptibility to complement-dependent killing, increased bacterial adhesion and invasion capacities to Vero cells, decreased blood bacterial loads, and attenuated virulence in infected ducks, when compared to the wild-type strain CH3. Moreover, RNA-Seq and real-time polymerase chain reaction analyses indicated that two genes were up-regulated and two were down-regulated in the mutant RA1062 genome. Furthermore, an animal protection experiment showed that immunization of ducks with inactivated RA1062 bacterin conferred effective cross-protection against challenge with the virulent R. anatipestifer serotypes 1, 2, and 10. This study presents evidence that the M949_RS01035 gene is involved in bacterial phenotype, virulence, and gene regulation in R. anatipestifer. The mutant strain RA1062 could be used as a cross-protective vaccine candidate.

Identification of DLD, by immunoproteomic analysis and evaluation as a potential vaccine antigen against three Vibrio species in Epinephelus coioides

Vibrio spp. represent a serious threat to the culture of Epinephelus coioides (Orange-spotted Grouper) in Southeast Asia. In this study we used two-dimensional electrophoresis (2-DE) and Western blotting to identify common immunogenic proteins of Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus. Membranes were probed with orange-spotted grouper anti-V. alginolyticus sera and accordingly 60, 58 and 48 immunogenic protein spots were detected. By matching analysis for the three Western blotting membranes, 6 cross immunogenic spots for the three Vibrio species were identified. They were Outer membrane protein W (OmpW), dihydrolipoamide dehydrogenase (DLD), succinate dehydrogenase flavoprotein subunit(SDHA), elongation factor Ts(Ts), peptide ABC transporter periplasmic peptide-binding protein and phosphoenolpyruvate carboxykinase(PEPCK). One of the proteins, DLD, was used to evaluate the cross protective function for E. coioides with a bacterial immunization and challenge method. The relative percent survival rate of E. coioides against V. alginolyticus, V. harveyi and V. parahaemolyticus was 90%, 86% and 80%, respectively. This work may provide potential cross protective vaccine candidate antigens for three Vibrio species, and DLD may be considered as an effective cross-protective immunogen against three Vibrio species.

Protein targets for broad‐spectrum mycosis vaccines: quantitative proteomic analysis of Aspergillus and Coccidioides and comparisons with other fungal pathogens

Aspergillus species are responsible for most cases of fatal mold infections in immunocompromised patients, particularly in those receiving hematopoietic stem cell transplants. Experimental vaccines in mouse models have demonstrated a promising avenue of approach for the prevention of aspergillosis, as well as infections caused by other fungal pathogens, such as Coccidioides, the etiological agent of valley fever (coccidioidomycosis). Here, we investigated the hyphal proteomes of Aspergillus fumigatus and Coccidioides posadasii via quantitative MS(E) mass spectrometry with the objective of developing a vaccine that cross-protects against these and other species of fungi. Several homologous proteins with highly conserved sequences were identified and quantified in A. fumigatus and C. posadasii. Many abundant proteins from the cell wall of A. fumigatus present themselves as possible cross-protective vaccine candidates, due to the high degree of sequence homology to other medically relevant fungal proteins and low homologies to human or murine proteins.

Immunoproteomics selection of cross-protective vaccine candidates from Riemerella anatipestifer serotypes 1 and 2

Riemerella anatipestifer (RA) is one of the most important bacterial pathogens of ducks and other avian species worldwide. Current approaches for controlling RA are hindered by the absence of effective vaccines, particularly cross-protective vaccines. In this present study, an immunoproteomics approach was used to identify cross-protective vaccine candidates against RA serotype 1 strain RAf63 and serotype 2 strain RAf153. First, whole-cell and secreted proteins of RAf153 and RAf63 were separated by two-dimensional gel electrophoresis. Then, western blotting of the proteome was performed using duck convalescent serum against RAf153, followed by matrix-assisted laser desorption/ionization time of flight mass spectrometry and peptide mass fingerprinting. Finally, common immunoreactive proteins from RAf153 and RAf63 were identified as cross-protective vaccine candidates, and so these were cloned and expressed recombinantly. The cross-protection abilities of purified recombinant protein vaccines were tested against homologous and heterologous virulent strains in a challenge model that followed vaccination. Six proteins were identified as cross-protective vaccine candidates. Three of these proteins showed reactivity with convalescent sera after prokaryotic expression, and the recombinant outer membrane protein A (OmpA) showed high protective indices against challenges with RAf153 (60%) and RAf63 (50%). In summary, we have developed a high-throughout, accurate, rapid and efficient method for the successful selection of cross-protective vaccine candidates.

Identification of conserved antigens from staphylococcal and streptococcal pathogens

The design of vaccines containing epitopes shared between different human pathogens may lead to cross-species protection. In order to identify potentially conserved bacterial antigens, bacteriophage expression libraries of genomic DNA from Streptococcus agalactiae, Streptococcus pneumoniae and Streptococcus pyogenes were probed with human sera from Staphylococcus aureus-infected and healthy individuals. By comparison with previous screening data from Staphylococcus epidermidis and Staph. aureus, putative antigenic, conserved domains across the genera were identified. In particular, three potentially antigenic conserved regions were identified based on the N-terminal domain of SACOL0609 (SdrD), the C-terminal domain of SACOL0723 (ScaB) and the C-terminus of SACOL1140 (IsdA) from Staph. aureus. The three domains were overexpressed, recombinant proteins were purified and polyclonal antisera raised against them recognized cell surface-located proteins from both staphylococcal and streptococcal species. The antisera were also able to opsonize both Staph. aureus and Strep. agalactiae thereby increasing their phagocytic uptake by human neutrophils. The conserved antigenic domains therefore represent potential cross-protective vaccine candidates.

Subtype prevalence, plasmid profiles and growing fluoroquinolone resistance in Shigella from Kolkata, India (2001-2007): a hospital-based study

Shigellosis is a major public health problem, and increasing antimicrobial resistance has complicated its treatment. We report isolation frequency, plasmid profiles and antimicrobial resistance of Shigella subtypes in Kolkata, India, from a prospective hospital-based study. Fresh stool or rectal swabs were collected from children (<5 years) attending the Diarrhea Treatment Unit of a governmental paediatric referral hospital in Kolkata. Samples were processed following standard methods over a 7-year period from January 2001 to December 2007. Of 4478 samples collected, 516 (11.5%) were positive for Shigella spp. S. flexneri (312; 6.9%) was the most frequently isolated serogroup, ranking before S. sonnei (123, 2.7%), S. dysenteriae (48, 1.1%) and S. boydii (33, 0.7%). Although 10 subtypes of S. flexneri were identified, the common ones circulating locally were S. flexneri 2a (179), S. flexneri 6 (38) and S. flexneri 3a (36). Knowledge of Shigella subtypes is important for vaccine development. The majority of Shigella isolates (81.0%) were multidrug (two or more antimicrobial classes) resistant and showed high minimum inhibitory concentration (MIC) with commonly used drugs like ampicillin, tetracycline, co-trimoxazole and nalidixic acid. Emergence of fluoroquinolone (FQ)-resistant S. dysenteriae type 1 (100.0%) in 2002-2003 was followed by frequent isolation (>25.0%) of FQ-resistant S. flexneri 2a, and S. flexneri 3a in 2004, which restricted use of fluoroquinolones for treatment. A number of smaller plasmids (<20 kb) with distinct patterns have been observed for several years in predominant subtypes. Long-term surveillance of Shigellae and their antimicrobial resistance are mandatory in endemic areas to formulate treatment policy until any suitable candidate vaccine is available to control the disease.

Gene Expression Profiling-BasedIn SilicoApproach to Identify Potential Vaccine Candidates and Drug Targets againstB. pertussisandB. parapertussis

Whooping cough (pertussis) caused by B. pertussis (B.p) is still serious public health threat. B. parapertussis (B.pp), closely related to B.p, also causes whooping cough. The incidence of B.pp infections has been increasing over the last decades, partly because pertussis vaccines have low efficiency against B.pp infections. Moreover, because the majority of pertussis patients are infants, common antimicrobial agents producing serious adverse reactions in infants are not fully satisfactory. Therefore, we try to identify potential vaccine candidates and alternative drug targets against both B.p and B.pp. This preliminary work integrates several different kinds of data from in silico analysis, comparative genomic hybridization, global transcriptional profiling, and protein-protein interaction (PPI) network to screen potential vaccine candidates and drug targets against the two species. Finally, 191 potential crossprotective vaccine candidates are identified. They have high transcriptional levels in both species, or are associated with virulence and pathogenesis. Moreover, these proteins are not only potentially surface-exposed in the bacteria, but also well conserved among the 165 B.p and B.pp strains. Among them, 22 candidates with high essentiality in the two PPI networks of B.p and B.pp are regarded as suitable drug targets against the two species. We just selected Bordetella as an example to develop a rapid and reliable approach for screening alternative drug targets that associated with novel protein pathways, complexes, and cellular functions against these antibiotic-resistant pathogens. Further researches focusing on the 191 vaccine candidates could accelerate the development of more effective vaccines and drug therapy against B.p and B.pp infection.