Synthesis and in vivo studies of carbohydrate-based vaccines against group A streptococcus

Carbohydrates, as carriers, providing numerous attachment points for the conjugation of peptide antigens and their optimal orientation for the recognition by cells of the immune system, reducing degradation of the attached peptide antigens and many other advantages make carbohydrate-based vaccine highly promising approach. Multiple copies of a single group A streptococcal (GAS) M protein derived specific peptide antigens (J8 or J14) were coupled onto carbohydrate cores (D-glucose and D-galactose) linked to lipophilic amino acids to produce a self-adjuvanting liposaccharide vaccine against GAS strains. In vivo experiments showed high serum IgG antibody titers against each of the incorporated peptide epitopes, J8 or J14.     

Influenza virus-like particles containing M2 induce broadly cross protective immunity

Background

Current influenza vaccines based on the hemagglutinin protein are strain specific and do not provide good protection against drifted viruses or emergence of new pandemic strains. An influenza vaccine that can confer cross-protection against antigenically different influenza A strains is highly desirable for improving public health.

Methodology/Principal Findings

To develop a cross protective vaccine, we generated influenza virus-like particles containing the highly conserved M2 protein in a membrane-anchored form (M2 VLPs), and investigated their immunogenicity and breadth of cross protection. Immunization of mice with M2 VLPs induced anti-M2 antibodies binding to virions of various strains, M2 specific T cell responses, and conferred long-lasting cross protection against heterologous and heterosubtypic influenza viruses. M2 immune sera were found to play an important role in providing cross protection against heterosubtypic virus and an antigenically distinct 2009 pandemic H1N1 virus, and depletion of dendritic and macrophage cells abolished this cross protection, providing new insight into cross-protective immune mechanisms.

Conclusions/Significance

These results suggest that presenting M2 on VLPs in a membrane-anchored form is a promising approach for developing broadly cross protective influenza vaccines.     

Nonimmunodominant regions are effective as building blocks in a streptococcal fusion protein vaccine

Identification of antigens that elicit protective immunity is essential for effective vaccine development. We investigated the related surface proteins of group B Streptococcus, Rib and alpha, as potential vaccine candidates. Paradoxically, nonimmunodominant regions proved to be of particular interest as vaccine components. Mouse antibodies elicited by Rib and alpha were directed almost exclusively against the C-terminal repeats and not against the N-terminal regions. However, a fusion protein derived from the nonimmunodominant N-terminal regions of Rib and alpha was much more immunogenic than one derived from the repeats and was immunogenic even without adjuvant. Moreover, antibodies to the N-terminal fusion protein protected against infection and inhibited bacterial invasion of epithelial cells. Similarly, the N-terminal region of Streptococcus pyogenes M22 protein, which is targeted by opsonic antibodies, is nonimmunodominant. These data indicate that nonimmunodominant regions of bacterial antigens could be valuable for vaccine development.     

Binding of human plasma proteins to Streptococcus pyogenes M protein determines the location of opsonic and non-opsonic epitopes

Antibodies directed against a pathogenic microorganism may recognize either protective or non-protective epitopes. Because antibodies elicited by a vaccine must be directed against protective epitopes, it is essential to understand the molecular properties that distinguish the two types of epitope. Here we analyse this problem for the antiphagocytic M protein of Streptococcus pyogenes, using the opsonizing capacity of antibodies to estimate their ability to confer protection in vivo. Our studies were focused on the M5 protein, which has three surface-exposed regions: the amino-terminal hypervariable region (HVR) and the B- and C-repeat regions. We first analysed the role of different M5 regions in phagocytosis resistance under non-immune conditions, employing chromosomal mutants expressing M5 proteins with internal deletions, and demonstrate that only the B-repeat region is essential for phagocytosis resistance. However, only antibodies to the HVR were opsonic. This apparent paradox could be explained by the ability of fibrinogen and albumin to specifically bind to the B- and C-repeats, respectively, causing inhibition of antibody binding under physiological conditions, while antibodies to the HVR could bind and promote deposition of complement. These data indicate that binding of human plasma proteins plays an important role in determining the location of opsonic and non-opsonic epitopes in streptococcal M protein.     

Self-assembly of trimethyl chitosan and poly(anionic amino acid)-peptide antigen conjugate to produce a potent self-adjuvanting nanovaccine delivery system

Short peptides derived from virulent pathogen proteins are promising antigens for the development of vaccines against infectious diseases. However, in order to mimic the danger signals associated with natural infection and stimulate an adaptive immune response, peptide antigens must be co-delivered with immune adjuvants. In this study, a group A streptococcus (GAS) M-protein derived B-cell epitope: J8, and universal T-helper epitope P25 containing peptides, were chemically coupled with different anionic amino acid-based polymers. The poly(anionic amino acid)-peptide antigen conjugates were mixed with trimethyl chitosan (TMC) to produce self-adjuvanting nanoparticulate vaccine candidates. TMC from two different sources were used to analyse their effect on immunogenicity. The nanoparticles produced from a peptide modified with 10 residues of polyglutamic acid and fungal TMC (NP5) stimulated production of the highest levels of serum antibodies in outbred mice. These antibodies were opsonic against all clinical GAS isolates tested.     

A dual-adjuvanting strategy for peptide-based subunit vaccines against group A Streptococcus: Lipidation and polyelectrolyte complexes

In order to improve the immunogenicity of peptide-based vaccines against group A Streptococcus (GAS), lipid moieties (C16 lipoamino acid and cholic acid) were conjugated with peptide antigen (P25-J8) and further modified with α-poly(glutamic acid) (α-PGA). Thus, positively charged lipopeptide vaccine candidates LCP-1 (P25-K(J8)-SS-C16-C16) and LCP-2 (P25-K(J8)-SS-K(cholic acid)) were synthesized. Negatively charged LCP-3 (P25-K(PGA-J8)-SS-K(cholic acid)) was also produced by attaching α-PGA to the J8 N-terminus of LCP-2. Polyelectrolyte complex (PEC) nanoparticles were formulated with heparin and/or trimethyl chitosan (TMC) for delivery of the lipopeptide vaccine candidates. The ability of the antigen-loaded nanoparticles to induce humoral immune responses was examined in outbred female Swiss mice following intranasal immunization. The antibodies produced were opsonic against all clinical GAS isolates tested.     

M protein typing of Thai group A streptococcal isolates by PCR-Restriction fragment length polymorphism analysis

Background  

Group A streptococcal (GAS) infections can lead to the development of severe post-infectious sequelae, such as rheumatic fever (RF) and rheumatic heart disease (RHD). RF and RHD are a major health concern in developing countries, and in indigenous populations of developed nations. The majority of GAS isolates are M protein-nontypeable (MNT) by standard serotyping. However, GAS typing is a necessary tool in the epidemiologically analysis of GAS and provides useful information for vaccine development. Although DNA sequencing is the most conclusive method for M protein typing, this is not a feasible approach especially in developing countries. To overcome this problem, we have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based assay for molecular typing the M protein gene (emm) of GAS.     

Immune response to superoxide dismutase in group A streptococcal infection

Extracellular localisation of manganese-dependent superoxide dismutase (SodA) by group A streptococcus (GAS) may have a role in protection of this pathogenic bacterium from exogenously produced reactive oxygen species. In this study we show that SodA is found both in surface protein extracts and in culture supernatants of GAS. To investigate whether SodA is a possible vaccine candidate outbred Quackenbush mice were subcutaneously vaccinated with recombinant SodA. Strong antibody responses which were moderately opsonic were elicited. These antibodies were unable to protect mice from intraperitoneal challenge with M1 GAS. We also show that SodA and p145 (a conserved peptide from the M-protein) antibodies are present at significantly higher levels amongst patients with rheumatic heart disease than in control subjects from the same endemic region. The higher SodA antibody levels in patients may be indicative of a role for this protein in pathogenesis of rheumatic heart disease but are more likely to be a marker of recent or recurrent streptococcal infection.     

FbaA- and M protein-based multi-epitope vaccine elicits strong protective immune responses against group A streptococcus in mouse model

We report the construction of a recombinant multivalent vaccine against group A streptococcus (GAS), designated F7M5. It contains seven predominant epitopes of FbaA identified by phage display technology, five non-tissue cross-reactive M protein fragments expressed on four selected serotypes prevalent in China, a Trojan antigen (TA) and a poly-alanine DR epitope (PADRE). BALB/c mice were immunized subcutaneously with F7M5 formulated with Freund's adjuvant, using recombinant FbaA and M protein in parallel as control. Using enzyme-linked immunosorbent assay (ELISA), mouse immune sera were assayed for IgG titers, IgG subclasses, and binding of F7M5 with M1GAS. Results indicated that the multivalent vaccine was highly immunogenic and elicited a balanced IgG1/IgG2a response. We also tested the reactivity of F7M5 to antistreptolysin O (ASO) antibodies in sera of GAS-infected patients and found a 95.8% positive rate, indicating that the epitopes of the vaccine were widely expressed in the prevalent serotypes of GAS. More importantly, the F7M5 vaccine elicited strong protective immune responses against lethal-dose challenge with a survival rate of 90%, but induced no cross-reactions or pathological lesions in mouse model, suggesting that F7M5 can be further developed as an effective and safe anti-GAS vaccine.     

Expression of different group A streptococcal M proteins in an isogenic background demonstrates diversity in adherence to and invasion of eukaryotic cells

The M protein of group A streptococcus (GAS) is considered to be a major virulence factor because it renders GAS resistant to phagocytosis and allows bacterial growth in human blood. There are more than 80 known serotypes of M proteins, and protective opsonic antibodies produced during disease in humans are serotype specific. M proteins also mediate bacterial adherence to epithelial cells of skin and pharynx. GAS strains vary in the genomic organization of the mga regulon, which contains the genes encoding M and M-like proteins and other virulence factors. This diversity of organization makes it difficult to assess virulence of M proteins of different serotypes, unless they can be expressed in an isogenic background. Here, we express M proteins of different serotypes in the M protein- and protein F1-deficient GAS strain, SAM2, which also lacks M-like proteins. Genes encoding M proteins of different serotypes (emmXs) have been integrated into the SAM2 chromosome in frame with the emm6.1 promoter and its mga regulon, resulting in similar levels of emmX expression. Although SAM2 exhibits a very low level of adherence to and invasion of HEp-2 and HaCaT cells, a SAM2-derived strain expressing M6 protein adheres to and invades both cell types. In contrast, the isogenic strain expressing M18 protein adheres to both cell types, but invades with a very low efficiency. A strain expressing M3 protein adheres to both types of cells, but its invasion of HEp-2 cells is serum dependent. A GAS strain expressing M6 protein does not compete with the isogenic strain expressing M18 protein for adherence to or invasion of HaCaT cells. We conclude that M proteins of different serotypes recognize different repertoires of receptors on the surfaces of eukaryotic cells.     

New multi-determinant strategy for a group A streptococcal vaccine designed for the Australian Aboriginal population

Infection with group A streptococci can result in acute and post-infectious pathology, including rheumatic fever and rheumatic heart disease. These diseases are associated with poverty and are increasing in incidence, particularly in developing countries and amongst indigenous populations, such as Australia's Aboriginal population, who suffer the highest incidence worldwide. Immunity to group A streptococci is mediated by antibodies against the M protein, a coiled-coil alpha helical surface protein of the bacterium. Vaccine development faces two substantial obstacles. Although opsonic antibodies directed against the N terminus of the protein are mostly responsible for serotypic immunity, more than 100 serotypes exist. Furthermore, whereas the pathogenesis of rheumatic fever is not well understood, increasing evidence indicates an autoimmune process. To develop a suitable vaccine candidate, we first identified a minimum, helical, non-host-cross-reactive peptide from the conserved C-terminal half of the protein and displayed this within a non-M-protein peptide sequence designed to maintain helical folding and antigenicity, J14 (refs. 8,9). As this region of the M protein is identical in only 70% of group A streptococci isolates, the optimal candidate might consist of the conserved determinant with common N-terminal sequences found in communities with endemic group A streptococci. We linked seven serotypic peptides with J14 using a new chemistry technique that enables the immunogen to display all the individual peptides pendant from an alkane backbone. This construct demonstrated excellent immunogenicity and protection in mice.     

Sublingual immunization with M2-based vaccine induces broad protective immunity against influenza

Background

The ectodomain of matrix protein 2 (M2e) of influenza A virus is a rationale target antigen candidate for the development of a universal vaccine against influenza as M2e undergoes little sequence variation amongst human influenza A strains. Vaccine-induced M2e-specific antibodies (Abs) have been shown to display significant cross-protective activity in animal models. M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study, we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored.

Methods and Results

A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs.

Conclusions

The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.     

Vaccination with M2e-based multiple antigenic peptides: characterization of the B cell response and protection efficacy in inbred and outbred mice

Background

The extracellular domain of the influenza A virus protein matrix protein 2 (M2e) is remarkably conserved between various human isolates and thus is a viable target antigen for a universal influenza vaccine. With the goal of inducing protection in multiple mouse haplotypes, M2e-based multiple antigenic peptides (M2e-MAP) were synthesized to contain promiscuous T helper determinants from the Plasmodium falciparum circumsporozoite protein, the hepatitis B virus antigen and the influenza virus hemagglutinin. Here, we investigated the nature of the M2e-MAP-induced B cell response in terms of the distribution of antibody (Ab) secreting cells (ASCs) and Ab isotypes, and tested the protective efficacy in various mouse strains.

Methodology/Principal Findings

Immunization of BALB/c mice with M2e-MAPs together with potent adjuvants, CpG 1826 oligonucleotides (ODN) and cholera toxin (CT) elicited high M2e-specific serum Ab titers that protected mice against viral challenge. Subcutaneous (s.c.) and intranasal (i.n.) delivery of M2e-MAPs resulted in the induction of IgG in serum and airway secretions, however only i.n. immunization induced anti-M2e IgA ASCs locally in the lungs, correlating with M2-specific IgA in the bronchio-alveolar lavage (BAL). Interestingly, both routes of vaccination resulted in equal protection against viral challenge. Moreover, M2e-MAPs induced cross-reactive and protective responses to diverse M2e peptides and variant influenza viruses. However, in contrast to BALB/c mice, immunization of other inbred and outbred mouse strains did not induce protective Abs. This correlated with a defect in T cell but not B cell responsiveness to the M2e-MAPs.

Conclusion/Significance

Anti-M2e Abs induced by M2e-MAPs are highly cross-reactive and can mediate protection to variant viruses. Although synthetic MAPs are promising designs for vaccines, future constructs will need to be optimized for use in the genetically heterogeneous human population.     

Effect of challenge of pigs previously immunised with inactivated vaccines containing homologous and heterologous <Emphasis Type="Italic">Mycoplasma hyopneumoniae</Emphasis> strains

Background  

Mycoplasma hyopneumoniae is the primary cause of enzootic pneumonia in pigs. Although vaccination is an important control tool, the results observed under field conditions are variable. This may be due to antigenic differences between the strains circulating in pig herds and the vaccine strain. This study compared the protective efficacy of four bacterins against challenge infection with a highly virulent field strain of M. hyopneumoniae.     

Nucleotide substitutions and small-scale insertion produce size and antigenic variation in group A streptococcal M1 protein

The presence of M protein on the surface of group A streptococci (GAS) confers the ability of the cell to resist phagocytosis in the absence of type-specific antibodies. It undergoes antigenic variation with more than 80 different serotypes having been defined. We have sequenced the M protein gene (emm1.1) from strain CS190 and present evidence that individual nucleotide substitutions are responsible for sequence variation in the N-terminal non-repeat region of emm1.1 and these substitutions have altered antibody recognition of opsonic epitopes. The N-terminal non-repeat domains of two other closely related strains, 71-155 and 76-088, were found to have sequence identical to emm1.1 with the addition of a 21 bp insert. This study provides the first evidence that nucleotide substitutions and small insertions are responsible for size and antigenic variation in the N terminal non-repeat domain of the M protein of GAS.     

The association of Duffy binding protein region II polymorphisms and its antigenicity in Plasmodium vivax isolates from Thailand

Plasmodium vivax Duffy binding protein II (DBPII) plays an important role in reticulocyte invasion and is a potential vaccine candidate against vivax malaria. However, polymorphisms in DBPII are a challenge for the successful design of a broadly protective vaccine. In this study, the genetic diversity of DBPII among Thai isolates was analyzed from Plasmodium vivax-infected blood samples and polymorphism characters were defined with the MEGA4 program. Sequence analysis identified 12 variant residues that are common among Thai DBPII haplotypes with variant residues L333F, L424I, W437R and I503K having the highest frequency. Variant residue D384K occurs in combination with either E385K or K386N/Q. Additionally, variant residue L424I occurs in conjunction with W437R in most Thai DBPII alleles and these variants frequently occur in combination with the I503K variant. The polymorphic patterns of Thai isolates were defined into 9 haplotypes (Thai DBL-1, -2, -3, etc.…). Thai DBL-2, -5, -6 haplotypes are the most common DBPII variants in Thai residents. To study the association of these Thai DBPII polymorphisms with antigenic character, the functional inhibition of anti-DBPII monoclonal antibodies against a panel of Thai DBL variants was characterized by an in vitro erythrocyte binding inhibition assay. The functional inhibition of anti-DBPII monoclonal antibodies 3C9, 2D10 and 2C6 against Thai variants was significantly different, suggesting that polymorphisms of Thai DBPII variants alter the antigenic character of the target epitopes. In contrast, anti-DBPII monoclonal antibody 2H2 inhibited all Thai DBPII variants equally well. Our results suggest that the immune efficacy of a DBPII vaccine will depend on the specificity of the anti-DBPII antibodies induced and that it is preferable to optimize responses to conserved epitopes for broadly neutralizing protection against P. vivax.     

Lateral gene transfer of streptococcal ICE element RD2 (region of difference 2) encoding secreted proteins

Background  

The genome of serotype M28 group A Streptococcus (GAS) strain MGAS6180 contains a novel genetic element named Region of Difference 2 (RD2) that encodes seven putative secreted extracellular proteins. RD2 is present in all serotype M28 strains and strains of several other GAS serotypes associated with female urogenital infections. We show here that the GAS RD2 element is present in strain MGAS6180 both as an integrative chromosomal form and a circular extrachromosomal element. RD2-like regions were identified in publicly available genome sequences of strains representing three of the five major group B streptococcal serotypes causing human disease. Ten RD2-encoded proteins have significant similarity to proteins involved in conjugative transfer of Streptococcus thermophilus integrative chromosomal elements (ICEs).     

Comparative Analysis of the Immunogenicity and Protective Effects of Inactivated EV71 Vaccines in Mice

Background

Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD). Three inactivated EV71 whole-virus vaccines of different strains developed by different manufacturers in mainland China have recently entered clinical trials. Although several studies on these vaccines have been published, a study directly comparing the immunogenicity and protective effects among them has not been carried out, which makes evaluating their relative effectiveness difficult. Thus, properly comparing newly developed vaccines has become a priority, especially in China.

Methods and Findings

This comparative immunogenicity study was carried out on vaccine strains (both live and inactivated), final container products (FCPs) without adjuvant, and corresponding FCPs containing adjuvant (FCP-As) produced by three manufacturers. These vaccines were evaluated by neutralizing antibody (NAb) responses induced by the same or different dosages at one or multiple time points post-immunization. The protective efficacy of the three vaccines was also determined in one-day-old ICR mice born to immunized female mice. Survival rates were observed in these suckling mice after challenge with 20 LD₅₀ of EV71/048M3C2. Three FCP-As, in a dose of 200 U, generated nearly 100% NAb positivity rates and similar geometric mean titers (GMTs), especially at 14–21 days post-inoculation. However, the dynamic NAb responses were different among three vaccine strains or three FCPs. The FCP-As at the lowest dose used in clinical trials (162 U) showed good protective effects in suckling mice against lethal challenge (90–100% survival), while the ED₅₀ of NAb responses and protective effects varied among three FCP-As.

Conclusions

These studies establish a standard method for measuring the immunogenicity of EV71 vaccines in mice. The data generated from our mouse model study indicated a clear dose-response relationship, which is important for vaccine quality control and assessment, especially for predicting protective efficacy in humans when combined with future clinical trial results.     

Application of the C3-Binding Motif of Streptococcal Pyrogenic Exotoxin B to Protect Mice from Invasive Group A Streptococcal Infection

Group A streptococcus (GAS) is an important human pathogen that produces several extracellular exotoxins to facilitate invasion and infection. Streptococcal pyrogenic exotoxin B (SPE B) has been demonstrated to be an important virulence factor of GAS. Our previous studies indicate that SPE B cleaves complement 3 (C3) and inhibits the activation of complement pathways. In this study, we constructed and expressed recombinant fragments of SPE B to examine the C3-binding site of SPE B. Using enzyme-linked immunosorbent assays and pull-down assays, we found that the C-terminal domain, containing amino-acid residues 345–398, of SPE B was the major binding site of human serum C3. We further identified a major, Ala³⁷⁶-Pro³⁹⁸, and a minor C3-binding motif, Gly³⁴⁶-Gly³⁶⁰, that both mediated the binding of C3 complement. Immunization with the C3-binding motifs protected mice against challenge with a lethal dose of non-invasive M49 strain GAS but not invasive M1 strains. To achieve higher efficiency against invasive M1 GAS infection, a combination of synthetic peptides derived from C-terminal epitope of streptolysin S (SLSpp) and from the major C3-binding motif of SPE B (PP6, Ala³⁷⁶-Pro³⁹⁸) was used to elicit specific immune response to those two important streptococcal exotoxins. Death rates and the severity of skin lesions decreased significantly in PP6/SLSpp-immunized mice that were infected with invasive M1 strains of GAS. These results indicate a combination of the C3-binding motif of SPE B and the protective epitope of SLS could be used as a subunit vaccine against invasive M1 strains group A streptococcal infection.