Immunobiology of the Classical Lancefield Group A Streptococcal Carbohydrate Antigen

Group A Streptococcus (GAS) is a preeminent human bacterial pathogen causing hundreds of millions of infections each year worldwide. In the clinical setting, the bacterium is easily identified by a rapid antigen test against the group A carbohydrate (GAC), a polysaccharide that comprises 30-50% of the GAS cell wall by weight. Originally described by Rebecca Lancefield in the 1930s, GAC consists of a polyrhamnose backbone and a N-acetylglucosamine (GlcNAc) side chain. This side chain, the species-defining immunodominant antigen, is potentially implicated in auto-reactive immune responses against human heart or brain tissue in post-streptococcal rheumatic fever or rheumatic heart disease. The recent discovery of the genetic locus encoding GAC biosynthesis and new insights into its chemical structure have provided novel insights into the assembly of the polysaccharide, its contribution to immune evasion and virulence, and ideas for safely harnessing its natural immunogenicity in vaccine design. This review serves to summarize the emerging new literature on GAC, the eponymous cell well antigen that provides structural integrity to GAS and directly interfaces with host innate and adaptive immune responses.

GroEL is an immunodominant surface-exposed antigen of Rickettsia typhi

Rickettsioses are neglected and emerging potentially fatal febrile diseases that are caused by obligate intracellular bacteria, rickettsiae. Rickettsia (R.) typhi and R. prowazekii constitute the typhus group (TG) of rickettsiae and are the causative agents of endemic and epidemic typhus, respectively. We recently generated a monoclonal antibody (BNI52) against R. typhi. Characterization of BNI52 revealed that it specifically recognizes TG rickettsiae but not the members of the spotted fever group (SFG) rickettsiae. We further show that BNI52 binds to protein fragments of ±30 kDa that are exposed on the bacterial surface and also present in the periplasmic space. These protein fragments apparently derive from the cytosolic GroEL protein of R. typhi and are also recognized by antibodies in the sera from patients and infected mice. Furthermore, BNI52 opsonizes the bacteria for the uptake by antigen presenting cells (APC), indicating a contribution of GroEL-specific antibodies to protective immunity. Finally, it is interesting that the GroEL protein belongs to 32 proteins that are differentially downregulated by R. typhi after passage through immunodeficient BALB/c CB17 SCID mice. This could be a hint that the rickettsia GroEL protein may have immunomodulatory properties as shown for the homologous protein from several other bacteria, too. Overall, the results of this study provide evidence that GroEL represents an immunodominant antigen of TG rickettsiae that is recognized by the humoral immune response against these pathogens and that may be interesting as a vaccine candidate. Apart from that, the BNI52 antibody represents a new tool for specific detection of TG rickettsiae in various diagnostic and experimental setups.

Identification of Potentially Therapeutic Immunogenic Peptides From Paracoccidioides lutzii Species

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America caused by the thermodimorphic fungi of the genus Paracoccidioides spp. Paracoccidioides lutzii (PL) is one of the 5 species that constitute the Paracoccidioides genus. PL expresses low amounts of glycoprotein (Gp) 43 (PLGp43) and PLGp43 displays few epitopes in common with the P. brasiliensis (PB) immunodominant antigen PBGp43, which is commonly used for serological diagnosis of PCM. This difference in structure between the glycoproteins markedly reduces the efficiency of serological diagnosis in patients infected with PL. We previously demonstrated that peptide 10 (P10) from the PBGp43 induces protective immune responses in in vitro and in vivo models of PB PCM. Since, P10 has proven to be a promising therapeutic to combat PB, we sought to identify peptides in PL that could similarly be applied for the treatment of PCM. PL yeast cell proteins were isolated from PL: dendritic cell co-cultures and subjected to immunoproteomics. This approach identified 18 PL peptides that demonstrated in silico predictions for immunogenicity. Eight of the most promising peptides were synthesized and applied to lymphocytes obtained from peptide-immunized or PL-infected mice as well as to in vitro cultures with peptides or dendritic cells pulsed the peptides. The peptides LBR5, LBR6 and LBR8 efficiently promoted CD4+ and CD8+ T cell proliferation and dendritic cells pulsed with LBR1, LBR3, LBR7 or LBR8 stimulated CD4+ T cell proliferation. We observed increases of IFN-γ in the supernatants from primed T cells for the conditions with peptides without or with dendritic cells, although IL-2 levels only increased in response to LBR8. These novel immunogenic peptides derived from PL will be employed to develop new peptide vaccine approaches and the proteins from which they are derived can be used to develop new diagnostic assays for PL and possibly other Paracoccidioides spp. These findings identify and characterize new peptides with a promising therapeutic profile for future against this important neglected systemic mycosis.

Dissecting serotype-specific contributions to live oral cholera vaccine efficacy

The O1 serogroup of Vibrio cholerae causes pandemic cholera and is divided into the Ogawa and Inaba serotypes. The O-antigen is V. cholerae’s immunodominant antigen, and the two serotypes, which differ by the presence or absence of a terminally methylated O-antigen, likely influence development of immunity to cholera and oral cholera vaccines (OCVs). However, there is no consensus regarding the relative immunological potency of each serotype, in part because previous studies relied on genetically heterogeneous strains. Here, we engineered matched serotype variants of a live OCV candidate, HaitiV, and used a germfree mouse model to evaluate the immunogenicity and protective efficacy of each vaccine serotype. By combining vibriocidal antibody quantification with single- and mixed-strain infection assays, we found that all three HaitiV variants—InabaV, OgawaV, and HikoV (bivalent Inaba/Ogawa)—were immunogenic and protective. None of the vaccine serotypes were superior across both of these vaccine metrics, suggesting that the impact of O1-serotype variation in OCV design, although detectable, is subtle. However, all three live vaccines significantly outperformed formalin-killed HikoV, supporting the idea that live OCV usage will bolster current cholera control practices. The potency of OCVs was found to be challenge strain-dependent, emphasizing the importance of appropriate strain selection for cholera challenge studies. Our findings and experimental approaches will be valuable for guiding the development of live OCVs and oral vaccines for additional pathogens.

Immunogenicity of Streptococcus equi subsp. equi recombinant SeM protein and bacterin in mice

ABSTRACT: The infection caused by Streptococcus equi, known as strangles, affects the respiratory system of horses, causing high morbidity and rapid spread among the herd. Bacterin vaccines, composed of inactivated whole cells of S. equi, have variable efficacy and duration. Infected animals produce specific antibodies against SeM, the immunodominant antigen of S. equi. This makes it a promising target for vaccine development. In this context, the objective of this work was to evaluate a vaccine combining S. equi bacterin and recombinant SeM protein. Mice were vaccinated with bacterin (S. equi ~1.2 × 108CFU/ml); rSeM protein (20μg); bacterin-rSeM combination; or PBS (Control Group) and challenged with a suspension of S. equi, containing 10 × LD50. All vaccinated mice survived the challenge and produced anti-rSeM and anti-S. equi antibodies, which were assessed by indirect ELISA. The Control Group reached endpoint criteria 96 h after infection. These results demonstrate that a vaccine combining the S. equi bacterin with rSeM protein protects mice against strangles. This combination vaccine could potentially protect horses and overcome the limitations of currently available strangle vaccines.

Dissecting serotype-specific contributions to live oral cholera vaccine efficacy

The O1 serogroup of Vibrio cholerae causes pandemic cholera and is divided into Ogawa and Inaba serotypes. The O-antigen is V. cholerae’s immunodominant antigen, and the two serotypes, which differ by the presence or absence of a terminally methylated O-antigen, likely influence development of immunity to cholera and oral cholera vaccines (OCVs). However, there is no consensus regarding the relative immunological potency of each serotype, in part because previous studies relied on genetically heterogenous strains. Here, we engineered matched serotype variants of a live OCV candidate, HaitiV, and used a germ-free mouse model to evaluate the immunogenicity and protective efficacy of each vaccine serotype. By combining vibriocidal antibody quantification with single and mixed strain infection assays, we found that all three HaitiV variants - InabaV, OgawaV, and HikoV (bivalent Inaba/Ogawa) - were immunogenic and protective, suggesting the impact of O1 serotype variation on OCV function may be minimal. The potency of OCVs was found to be challenge strain-dependent, emphasizing the importance of appropriate strain selection for cholera challenge studies. Our findings and experimental approaches will be valuable for guiding the development of live OCVs and oral vaccines for additional pathogens.

Psp140: an immunodominant antigen in the supernatant of Streptococcus pneumoniae culture

Background and Objectives: Streptococcus pneumoniae causes many lethal infections. Due to its reduced sensitivity to commonly used antibiotics, development of new strategies against pneumococcal infections seems to be necessary. We aimed to investigate immunodominant antigens in S. pneumoniae culture supernatant in order to develop novel targets for pneumococcal vaccines. Materials and Methods: In this study S. pneumoniae ATCC49619 was sub-cultured into BHI broth from overnight culture at 37°C for 4 h. The supernatant proteins were precipitated using acetone precipitation method. A rabbit was intramuscularly immunized with alum adjuvant and 100 μg pneumococcal supernatant proteins, 6 times at 14 days' intervals to produce hyperimmune serum. ELISA assay was performed to determine the antibody level response to pneumococcal secretory proteins. Then dot blot applied for rapid evaluation of hyperimmune serum reactivity to pneumococcus supernatant proteins. The western blot was also used to determine the interaction of supernatant proteins with immunogenic rabbit's hyperimmune-serum. Results: According to the western blot analysis, the immunodominant protein had 140KDa molecular weight and designated as pneumococcal secretory protein140 (Psp140). Conclusion: The Psp140 protein in the supernatant of S. pneumoniae culture is an immunodominant protein and it is likely related to pneumococcal secretory protein or surface exposed protein which released into culture supernatant during bacterial growth.

Identification of Protective Peptides of Fasciola Hepatica-Derived Cathepsin L1 (FhCL1) in Vaccinated Sheep by a Linear B-cell Epitope Mapping Approach

Background Fasciolosis is one of the most important parasitic diseases of livestock. The need for better control strategies gave rise to the identification of various vaccine candidates. The recombinant form of a member of the cysteine protease family, cathepsin L1 of Fasciola hepatica (FhCL1) has been a vaccine target for the past few decades since it has been shown to behave as an immunodominant antigen. However, when FhCL1 was used as vaccine, it has been observed to elicit significant protection in some trials, whereas no protection was provided in others.Methods In order to improve vaccine development strategy, we conducted a linear B-cell epitope mapping of FhCL1 in vaccinated-protected, vaccinated but not protected, and unvaccinated-infected sheep.Results Our study showed that the pattern and dynamic of peptide recognition varied noticeably between protected and non-protected animals, and that the regions 55–63 and 77–84, which are within the propeptide, and regions 102–114 and 265–273 of FhCL1 were specifically recognised only by vaccinated-protected animals with significant decrease in fluke burden. In addition, these animals also showed significant production of specific IgG2, whereas none was observed in non-protected and infected animals.Conclusions We have identified forty-two residues of FhCL1 that contributed to protective immunity against infection with F. hepatica in sheep. Our results provide indications in relation to key aspects of the immune response. Given the variable outcomes of vaccination trials conducted in ruminants to date, this study adds new insights to improve strategies of vaccine development.

Immunization with recombinant enolase of Sporothrix spp. (rSsEno) confers effective protection against sporotrichosis in mice

In recent years, research has focused on the immunoreactive components of the Sporothrix schenckii cell wall that can be relevant targets for preventive and therapeutic vaccines against sporotrichosis, an emergent worldwide mycosis. In a previous study, we identified a 47-kDa enolase as an immunodominant antigen in mice vaccinated with an adjuvanted mixture of S. schenckii cell wall proteins. Here, we sought to assess the protective potential of a Sporothrix spp. recombinant enolase (rSsEno) formulated with or without the adjuvant Montanide Pet-GelA (PGA) against the S. brasiliensis infection in mice. Mice that were immunized with rSsEno plus PGA showed increased antibody titters against rSsEno and increased median survival time when challenged with S. brasiliensis as compared with mice that had not been immunized or that were immunized with rSsEno alone. Immunization with rSsEno plus PGA induced a predominantly T-helper 1 cytokine pattern after in vitro stimulation of splenic cells with rSsEno: elevated levels of IFN-γ and IL-2, as well as of other cytokines involved in host defense against sporotrichosis, such as TNF-alpha, IL-6, and IL-4. Furthermore, we show for the first time the presence of enolase in the cell wall of both S. schenckii and S. brasiliensis. As a whole, our results suggest that enolase could be used as a potential antigenic target for vaccinal purposes against sporotrichosis.

Purification and Characterisation of Badger IgA and Its Detection in the Context of Tuberculosis

European badgers are a wildlife reservoir of bovine tuberculosis in parts of Great Britain. Accurate diagnosis of tuberculosis in badgers is important for the development of strategies for the control of the disease. Sensitive serological tests for badger TB are needed for reasons such as cost and simplicity. Assay of mucosal IgA could be useful for diagnosing respiratory pathogens such as Mycobacterium bovis and for monitoring the response to mucosal vaccination. To develop an IgA assay, we purified secretory IgA from badger bile, identifying secretory component (SC), heavy chain (HC) and light chain (LC), at 66, 46 and 27 Kda, respectively, on the basis of size comparison with other species. Monoclonal antibodies (mAbs) were generated to purified IgA. We selected two for ELISA development. The detection limit of the IgA-specific mAbs was found to be approximately 20 ng/mL when titrated against purified badger bile. One monoclonal antibody specific for badger IgA was used to detect IgA in serum and tracheal aspirate with specificity to an immunodominant antigen of M. bovis. An M. bovis infection dose-dependent IgA response was observed in experimentally infected badgers. IgA was also detected by immunohistochemistry in the lungs of bTB-infected badgers. With further characterisation, these represent new reagents for the study of the IgA response in badgers.