RESUMEN
Group A Streptococcus is a pathogen of global importance, but despite the ubiquity of group A Streptococcus infections, the relationship between infection, colonization, and immunity is still not completely understood. The M protein, encoded by the emm gene, is a major virulence factor and vaccine candidate and forms the basis of a number of classification systems. Longitudinal patterns of emm types collected from 457 Fijian schoolchildren over a 10-month period were analyzed. No evidence of tissue tropism was observed, and there was no apparent selective pressure or constraint of emm types. Patterns of emm type acquisition suggest limited, if any, modification of future infection based on infection history. Where impetigo is the dominant mode of transmission, circulating emm types either may not be constrained by ecological niches or population immunity to the M protein, or they may require several infections over a longer period of time to induce such immunity.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Portadoras/inmunología , Enfermedades Cutáneas Bacterianas/inmunología , Infecciones Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Adolescente , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Niño , Preescolar , Femenino , Fiji/epidemiología , Humanos , Estudios Longitudinales , Masculino , Enfermedades Cutáneas Bacterianas/epidemiología , Infecciones Estreptocócicas/epidemiología , EstudiantesRESUMEN
Peptides offer enormous promise as vaccines to prevent and protect against many infectious and noninfectious diseases. However, to date, limited vaccine efficacy has been reported and none have been licensed for human use. Innovative ways to enhance their immunogenicity are being tested, but rational sequence modification as a means to improve immune responsiveness has been neglected. Our objective was to establish a two-step generic protocol to modify defined amino acids of a helical peptide epitope to create a superior immunogen. Peptide variants of p145, a conserved helical peptide epitope from the M protein of Streptococcus pyogenes, were designed by exchanging one amino acid at a time, without altering their α-helical structure, which is required for correct antigenicity. The immunogenicities of new peptides were assessed in outbred mice. Vaccine efficacy was assessed in a skin challenge and invasive disease model. Out of 86 variants of p145, seven amino acid substitutions were selected and made the basis of the design for 18 new peptides. Of these, 13 were more immunogenic than p145; 7 induced Abs with significantly higher affinity for p145 than Abs induced by p145 itself; and 1 peptide induced more than 10,000-fold greater protection following challenge than the parent peptide. This peptide also only required a single immunization (compared with three immunizations with the parent peptide) to induce complete protection against invasive streptococcal disease. This study defines a strategy to rationally improve the immunogenicity of peptides and will have broad applicability to the development of vaccines for infectious and noninfectious diseases.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/metabolismo , Fragmentos de Péptidos/metabolismo , Infecciones Estreptocócicas/inmunología , Vacunas Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Humanos , Inmunidad Humoral , Inmunización , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Ingeniería de Proteínas , Infecciones Estreptocócicas/prevención & control , Vacunas de SubunidadRESUMEN
The immunobiology underlying the slow acquisition of skin immunity to group A streptococci (GAS), is not understood, but attributed to specific virulence factors impeding innate immunity and significant antigenic diversity of the type-specific M-protein, hindering acquired immunity. We used a number of epidemiologically distinct GAS strains to model the development of acquired immunity. We show that infection leads to antibody responses to the serotype-specific determinants on the M-protein and profound protective immunity; however, memory B cells do not develop and immunity is rapidly lost. Furthermore, antibodies do not develop to a conserved M-protein epitope that is able to induce immunity following vaccination. However, if re-infected with the same strain within three weeks, enduring immunity and memory B-cells (MBCs) to type-specific epitopes do develop. Such MBCs can adoptively transfer protection to naïve recipients. Thus, highly protective M-protein-specific MBCs may never develop following a single episode of pyoderma, contributing to the slow acquisition of immunity and to streptococcal endemicity in at-risk populations.
Asunto(s)
Memoria Inmunológica/inmunología , Piodermia/inmunología , Piodermia/microbiología , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Animales , Ensayo de Inmunoadsorción Enzimática , Ratones , Streptococcus pyogenesRESUMEN
Cluster of virulence responder/sensor (CovR/S) mutant group A streptococci (GAS) are serious human pathogens of multiple M protein strains that upregulate expression of virulence factors, including the IL-8 proteaseStreptococcus pyogenescell envelope proteinase (SpyCEP), thus blunting neutrophil-mediated killing and enabling ingress of bacteria from a superficial wound to deep tissue. We previously showed that a combination vaccine incorporating J8-DT (conserved peptide vaccine from the M protein) and a recombinant SpyCEP fragment protects against CovR/S mutants. To enhance the vaccine's safety profile, we identified a minimal epitope (S2) that was the target for anti-SpyCEP Abs that could protect IL-8 from SpyCEP-mediated proteolysis. Abs from healthy humans and from mice experimentally infected with GAS also recognized S2, albeit at low titers. Native SpyCEP may be poorly immunogenic (cryptic or subdominant), and it would be to the organism's advantage if the host did not induce a strong Ab response against it. However, S2 conjugated to diphtheria toxoid is highly immunogenic and induces Abs that recognize and neutralize SpyCEP. Hence, we describe a two-component peptide vaccine that induces Abs (anti-S2) that protect IL-8 from proteolysis and other Abs (anti-J8) that cause strain-independent killing in the presence of neutrophils. We show that either component alone is ineffectual in preventing skin infection and bacteremia due to CovR/S mutants but that the combination induces complete protection. This protection correlated with a significant influx of neutrophils to the infection site. The data strongly suggest that the lack of natural immunity to hypervirulent GAS strains in humans could be rectified by this combination vaccine.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Portadoras/inmunología , Péptido Hidrolasas/inmunología , Infecciones Estreptocócicas/inmunología , Vacunas Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Bacteriemia/inmunología , Bacteriemia/microbiología , Bacteriemia/prevención & control , Proteínas Bacterianas/inmunología , Toxoide Diftérico/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Neutrófilos/inmunología , Péptido Hidrolasas/biosíntesis , Péptido Hidrolasas/genética , Piel/microbiología , Enfermedades Cutáneas Bacterianas/inmunología , Enfermedades Cutáneas Bacterianas/microbiología , Enfermedades Cutáneas Bacterianas/prevención & control , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/prevención & control , Streptococcus pyogenes/patogenicidad , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/inmunología , Factores de Virulencia/biosíntesis , Factores de Virulencia/genética , Factores de Virulencia/inmunologíaRESUMEN
Four groupâ A streptococcal glycolipopeptide vaccine candidates with different lipidic adjuvanting moieties were prepared and characterized. The immunogenicity of the compounds was evaluated by macrophage and dendritic cell uptake studies and by in vivo quantification of systemic IgG antibody by ELISA. Three of the candidates showed significant induction of the IgG response.
Asunto(s)
Adyuvantes Inmunológicos/síntesis química , Inmunoglobulina G/sangre , Lípidos/inmunología , Vacunas Estreptocócicas/síntesis química , Vacunas Estreptocócicas/inmunología , Adyuvantes Inmunológicos/química , Animales , Ensayo de Inmunoadsorción Enzimática , Lípidos/síntesis química , Lípidos/química , Ratones , Tamaño de la Partícula , Péptidos/síntesis química , Péptidos/química , Péptidos/inmunología , Vacunas Estreptocócicas/químicaRESUMEN
Infections caused by Streptococcus pyogenes (group A Streptococcus [GAS]) are highly prevalent in the tropics, in developing countries, and in the Indigenous populations of developed countries. These infections and their sequelae are responsible for almost 500,000 lives lost prematurely each year. A synthetic peptide vaccine (J8-DT) from the conserved region of the M protein has shown efficacy against disease that follows i.p. inoculation of bacteria. By developing a murine model for infection that closely mimics human skin infection, we show that the vaccine can protect against pyoderma and subsequent bacteremia caused by multiple GAS strains, including strains endemic in Aboriginal communities in the Northern Territory of Australia. However, the vaccine was ineffective against a hypervirulent cluster of virulence responder/sensor mutant GAS strain; this correlated with the strain's ability to degrade CXC chemokines, thereby preventing neutrophil chemotaxis. By combining J8-DT with an inactive form of the streptococcal CXC protease, S. pyogenes cell envelope proteinase, we developed a combination vaccine that is highly effective in blocking CXC chemokine degradation and permits opsonic Abs to kill the bacteria. Mice receiving the combination vaccine were strongly protected against pyoderma and bacteremia, as evidenced by a 100-1000-fold reduction in bacterial burden following challenge. To our knowledge, a vaccine requiring Abs to target two independent virulence factors of an organism is unique.
Asunto(s)
Bacteriemia , Piodermia/prevención & control , Vacunas Estreptocócicas/inmunología , Streptococcus/inmunología , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Modelos Animales de Enfermedad , Femenino , Inmunidad Innata , Memoria Inmunológica , Macrófagos/inmunología , Ratones , Neutrófilos/inmunología , Péptidos/inmunología , Piodermia/patología , Proteínas Recombinantes/inmunología , Infecciones Cutáneas Estafilocócicas/patología , Infecciones Cutáneas Estafilocócicas/prevención & control , Vacunas Estreptocócicas/genética , Vacunas Sintéticas/genéticaRESUMEN
Infection with Burkholderia pseudomallei causes melioidosis, a disease with a high mortality rate (20% in Australia and 40% in Southeast Asia). Neurological melioidosis is particularly prevalent in northern Australian patients and involves brain stem infection, which can progress to the spinal cord; however, the route by which the bacteria invade the central nervous system (CNS) is unknown. We have previously demonstrated that B. pseudomallei can infect the olfactory and trigeminal nerves within the nasal cavity following intranasal inoculation. As the trigeminal nerve projects into the brain stem, we investigated whether the bacteria could continue along this nerve to penetrate the CNS. After intranasal inoculation of mice, B. pseudomallei caused low-level localized infection within the nasal cavity epithelium, prior to invasion of the trigeminal nerve in small numbers. B. pseudomallei rapidly invaded the trigeminal nerve and crossed the astrocytic barrier to enter the brain stem within 24 h and then rapidly progressed over 2,000 µm into the spinal cord. To rule out that the bacteria used a hematogenous route, we used a capsule-deficient mutant of B. pseudomallei that does not survive in the blood and found that it also entered the CNS via the trigeminal nerve. This suggests that the primary route of entry is via the nerves that innervate the nasal cavity. We found that actin-mediated motility could facilitate initial infection of the olfactory epithelium. Thus, we have demonstrated that B. pseudomallei can rapidly infect the brain and spinal cord via the trigeminal nerve branches that innervate the nasal cavity.
Asunto(s)
Tronco Encefálico/microbiología , Burkholderia pseudomallei/patogenicidad , Cavidad Nasal/microbiología , Médula Espinal/microbiología , Nervio Trigémino/microbiología , Administración Intranasal/métodos , Animales , Melioidosis/microbiología , RatonesRESUMEN
Melioidosis, caused by the bacterium Burkholderia pseudomallei, is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery of B. pseudomallei to establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improved B. pseudomallei survival in vivo and triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response to B. pseudomallei infection, we infected human olfactory ensheathing cells (OECs) in vitro and measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of the B. pseudomallei in a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I in B. pseudomallei survival in vivo following inhalation infection and the antibacterial signaling network that exists in human OM and OECs.
Asunto(s)
Cápsulas Bacterianas/inmunología , Burkholderia pseudomallei/inmunología , Interacciones Huésped-Patógeno/inmunología , Melioidosis/inmunología , Melioidosis/microbiología , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/microbiología , Animales , Cápsulas Bacterianas/genética , Carga Bacteriana , Burkholderia pseudomallei/genética , Células Cultivadas , Biología Computacional/métodos , Citocinas/metabolismo , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunidad Innata , Melioidosis/genética , Melioidosis/metabolismo , Ratones , Mutación , Infiltración Neutrófila , Neuronas Receptoras Olfatorias/inmunología , Neuronas Receptoras Olfatorias/metabolismo , Neuronas Receptoras Olfatorias/microbiología , Infecciones del Sistema Respiratorio/genética , Infecciones del Sistema Respiratorio/metabolismo , Transducción de Señal , Virulencia , Factores de VirulenciaRESUMEN
BACKGROUND: The spectrum of techniques to detect malaria parasites in whole blood is limited to measuring parasites in circulation. One approach that is currently used to enumerate total parasite bio-burden involves the use of bio-luminescent parasites. As an alternative approach, this study describes the use of a commercial ELISA human parasite lactate dehydrogenase (pLDH) detection kit to estimate total parasite bio-burden in murine malaria models. METHODS: The cross reactivity of pLDH in a commercial human malaria pLDH diagnostic kit was established in different components of blood for different murine malaria models. The use of pLDH as a measure of parasite bio-burden was evaluated by examining pLDH in relation to peripheral blood parasitaemia as determined by microscopy and calculating total parasite bio-burden using a bio-luminescent Plasmodium berghei ANKA luciferase parasite. RESULTS: The pLDH antigen was detected in all four murine Plasmodium species and in all components of Plasmodium-infected blood. A significant correlation (r = 0.6922, P value <0.0001) was observed between total parasite bio-burden, measured as log average radiance, and concentration of pLDH units. CONCLUSIONS: This high throughput assay is a suitable measure of total parasite bio-burden in murine malaria infections. Unlike existing methods, it permits the estimation of both circulating and sequestered parasites, allowing a more accurate assessment of parasite bio-burden.
Asunto(s)
L-Lactato Deshidrogenasa/sangre , Malaria/sangre , Malaria/diagnóstico , Plasmodium berghei/enzimología , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Parasitemia/sangre , Parasitemia/diagnóstico , Proteínas Protozoarias/sangreRESUMEN
Infection with Group A Streptococcus (GAS) can result in a range of different illnesses, some of which are fatal. Currently, our efforts to develop a vaccine against GAS focuses on the lipid core peptide (LCP) system, a subunit vaccine containing a lipoamino acid (LAA) moiety which allows the stimulation of systemic antibody activity. In the present study, a peptide (J14) representing the B-cell epitope from the GAS M protein was incorporated alongside a universal T-helper epitope (P25) in four LCP constructs of different spatial orientation or LAA lengths. Through structure-activity studies, it was discovered that while the alteration of the LCP orientation had a weaker effect on immunostimulation, increasing the LAA side chain length within the construct increased antibody responses in murine models. Furthermore, the mice immunised with the lead LCP construct were also able to maintain antibody activity throughout the course of five months. These findings highlight the importance of LAA moieties in the development of intranasal peptide vaccines and confirmed that its side chain length has an effect on the immunogenicity of the structure.
Asunto(s)
Vacunas Bacterianas/química , Vacunas Bacterianas/farmacología , Lípidos/química , Péptidos/química , Streptococcus pyogenes/inmunología , Secuencia de Aminoácidos , Animales , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Ratones , Microscopía Electrónica de Transmisión , Relación Estructura-ActividadRESUMEN
The brain is well protected against microbial invasion by cellular barriers, such as the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). In addition, cells within the central nervous system (CNS) are capable of producing an immune response against invading pathogens. Nonetheless, a range of pathogenic microbes make their way to the CNS, and the resulting infections can cause significant morbidity and mortality. Bacteria, amoebae, fungi, and viruses are capable of CNS invasion, with the latter using axonal transport as a common route of infection. In this review, we compare the mechanisms by which bacterial pathogens reach the CNS and infect the brain. In particular, we focus on recent data regarding mechanisms of bacterial translocation from the nasal mucosa to the brain, which represents a little explored pathway of bacterial invasion but has been proposed as being particularly important in explaining how infection with Burkholderia pseudomallei can result in melioidosis encephalomyelitis.
Asunto(s)
Infecciones del Sistema Nervioso Central/microbiología , Animales , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/microbiología , Infecciones del Sistema Nervioso Central/inmunología , Infecciones del Sistema Nervioso Central/transmisión , Humanos , Vigilancia Inmunológica , Cavidad Nasal/microbiología , Nervio Olfatorio/microbiología , Nervio Trigémino/microbiologíaRESUMEN
BACKGROUND: The ability to undertake controlled human malaria infection (CHMI) studies for preliminary evaluation of malaria vaccine candidates and anti-malaria drug efficacy has been limited by the need for access to sporozoite infected mosquitoes, aseptic, purified, cryopreserved sporozoites or blood-stage malaria parasites derived ex vivo from malaria infected individuals. Three different strategies are described for the manufacture of clinical grade cultured malaria cell banks suitable for use in CHMI studies. METHODS: Good Manufacturing Practices (GMP)-grade Plasmodium falciparum NF54, clinically isolated 3D7, and research-grade P. falciparum 7G8 blood-stage malaria parasites were cultured separately in GMP-compliant facilities using screened blood components and then cryopreserved to produce three P. falciparum blood-stage malaria cell banks. These cell banks were evaluated according to specific criteria (parasitaemia, identity, viability, sterility, presence of endotoxin, presence of mycoplasma or other viral agents and in vitro anti-malarial drug sensitivity of the cell bank malaria parasites) to ensure they met the criteria to permit product release according to GMP requirements. RESULTS: The P. falciparum NF54, 3D7 and 7G8 cell banks consisted of >78% ring stage parasites with a ring stage parasitaemia of >1.4%. Parasites were viable in vitro following thawing. The cell banks were free from contamination with bacteria, mycoplasma and a broad panel of viruses. The P. falciparum NF54, 3D7 and 7G8 parasites exhibited differential anti-malarial drug susceptibilities. The P. falciparum NF54 and 3D7 parasites were susceptible to all anti-malaria compounds tested, whereas the P. falciparum 7G8 parasites were resistant/had decreased susceptibility to four compounds. Following testing, all defined release criteria were met and the P. falciparum cell banks were deemed suitable for release. Ethical approval has been obtained for administration to human volunteers. CONCLUSIONS: The production of cultured P. falciparum blood-stage malaria cell banks represents a suitable approach for the generation of material suitable for CHMI studies. A key feature of this culture-based approach is the ability to take research-grade material through to a product suitable for administration in clinical trials.
Asunto(s)
Bancos de Muestras Biológicas , Ensayos Clínicos como Asunto , Malaria/tratamiento farmacológico , Plasmodium falciparum/crecimiento & desarrollo , Esporozoítos/crecimiento & desarrollo , Animales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Humanos , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/inmunología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/inmunología , Esporozoítos/efectos de los fármacos , Esporozoítos/inmunologíaRESUMEN
Streptococcus pyogenes (group A Streptococcus [GAS]) is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Vaccination with J8, a conserved region synthetic peptide derived from the M-protein of GAS and containing only 12 aa from GAS, when conjugated to diphtheria toxoid, has been shown to protect mice against a lethal GAS challenge. Protection has been previously shown to be Ab-mediated. J8 does not contain a dominant GAS-specific T cell epitope. The current study examined long-term Ab memory and dissected the role of B and T cells. Our results demonstrated that vaccination generates specific memory B cells (MBC) and long-lasting Ab responses. The MBC response can be activated following boost with Ag or limiting numbers of whole bacteria. We further show that these memory responses protect against systemic infection with GAS. T cell help is required for activation of MBC but can be provided by naive T cells responding directly to GAS at the time of infection. Thus, individuals whose T cells do not recognize the short synthetic peptide in the vaccine will be able to generate a protective and rapid memory Ab response at the time of infection. These studies significantly strengthen previous findings, which showed that protection by the J8-diphtheria toxoid vaccine is Ab-mediated and suggest that in vaccine design for other organisms the source of T cell help for Ab responses need not be limited to sequences from the organism itself.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/microbiología , Memoria Inmunológica/inmunología , Infecciones Estreptocócicas/prevención & control , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/microbiología , Vacunas de Subunidad/administración & dosificación , Animales , Anticuerpos Antibacterianos/administración & dosificación , Subgrupos de Linfocitos B/metabolismo , Inmunización Secundaria/métodos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Factores de Tiempo , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/uso terapéuticoRESUMEN
Subunit vaccines offer a means to produce safer, more defined vaccines compared to traditional whole microorganism approaches. Subunit antigens, however, exhibit weak immunity, which is normally overcome through coadministration with adjuvants. Enhanced vaccine properties (e.g., improved potency) can be obtained by linking antigen and adjuvant, as observed for synthetic peptide antigens and Toll-like receptor 2 (TLR2) ligands. As few protective peptide antigens have been reported, compared to protein antigens, we sought to extend the utility of this approach to recombinant proteins, while ensuring that conjugation reactions yielded a single, molecularly defined product. Herein we describe the development and optimization of techniques that enable the efficient, site-specific attachment of three synthetic TLR2 ligands (lipid core peptide (LCP), Pam2Cys, and Pam3Cys) onto engineered protein antigens, permitting the selection of optimal TLR2 agonists during the vaccine development process. Using this approach, broadly protective (J14) and population targeted (seven M protein N-terminal antigens) multiantigenic vaccines against group A streptococcus (GAS; Streptococcus pyogenes) were produced and observed to self-assemble in PBS to yield nanoparticules (69, 101, and 123 nm, respectively). All nanoparticle formulations exhibited self-adjuvanting properties, with rapid, persistent, antigen-specific IgG antibody responses elicited toward each antigen in subcutaneously immunized C57BL/6J mice. These antibodies were demonstrated to strongly bind to the cell surface of five GAS serotypes that are not represented by vaccine M protein N-terminal antigens, are among the top 20 circulating strains in developed countries, and are associated with clinical disease, suggesting that these vaccines may elicit broadly protective immune responses.
Asunto(s)
Nanopartículas/química , Vacunas Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 2/inmunología , Animales , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Nanopartículas/administración & dosificación , Tamaño de la Partícula , Vacunas Estreptocócicas/administración & dosificación , Vacunas Estreptocócicas/síntesis química , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
Effective and low-cost vaccines are essential to control severe group A streptococcus (GAS) infections prevalent in low-income nations and the Australian aboriginal communities. Highly diverse and endemic circulating GAS strains mandate broad-coverage and customized vaccines. This study describes an approach to deliver cross-reactive antigens from endemic GAS strains using modular virus-like particle (VLP) and capsomere systems. The antigens studied were three heterologous N-terminal peptides (GAS1, GAS2, and GAS3) from the GAS surface M-protein that are specific to endemic strains in Australia Northern Territory Aboriginal communities. In vivo data presented here demonstrated salient characteristics of the modular delivery systems in the context of GAS vaccine design. First, the antigenic peptides, when delivered by unadjuvanted modular VLPs or adjuvanted capsomeres, induced high titers of peptide-specific IgG antibodies (over 1 × 10(4) ). Second, delivery by capsomere was superior to VLP for one of the peptides investigated (GAS3), demonstrating that the delivery system relative effectiveness was antigen-dependant. Third, significant cross-reactivity of GAS2-induced IgG with GAS1 was observed using either VLP or capsomere, showing the possibility of broad-coverage vaccine design using these delivery systems and cross-reactive antigens. Fourth, a formulation containing three pre-mixed modular VLPs, each at a low dose of 5 µg (corresponding to <600 ng of each GAS peptide), induced significant titers of IgGs specific to each peptide, demonstrating that a multivalent, broad-coverage VLP vaccine formulation was possible. In summary, the modular VLPs and capsomeres reported here demonstrate, with promising preliminary data, innovative ways to design GAS vaccines using VLP and capsomere delivery systems amenable to microbial synthesis, potentially adoptable by developing countries.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Portadoras/inmunología , Vacunas Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Vacunas Sintéticas/inmunología , Vacunas de Virosoma/inmunología , Virosomas/metabolismo , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Portadoras/genética , Reacciones Cruzadas , Humanos , Inmunoglobulina G/sangre , Ratones , Nativos de Hawái y Otras Islas del Pacífico , Northern Territory/epidemiología , Infecciones Estreptocócicas/epidemiología , Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/administración & dosificación , Vacunas Estreptocócicas/genética , Vacunas Estreptocócicas/aislamiento & purificación , Streptococcus pyogenes/genética , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/aislamiento & purificación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/aislamiento & purificación , Vacunas de Virosoma/administración & dosificación , Vacunas de Virosoma/genética , Vacunas de Virosoma/aislamiento & purificación , Virosomas/genéticaRESUMEN
Peptides are of great interest to be used as vaccine antigens due to their safety, ease of manufacturing and specificity in generating immune response. There have been massive discoveries of peptide antigens over the past decade. However, peptides alone are poorly immunogenic, which demand co-administration with strong adjuvant to enhance their immunogenicity. Recently, fibril-forming peptides such as Q11 and lipoamino acid-based carrier have been identified to induce substantial immune responses when covalently linked to peptide epitope. In this study, we have incorporated either Q11 or lipoamino acids to a peptide epitope (J14) derived from M protein of group A streptococcus to develop self-adjuvanting vaccines. J14, Q11 and lipoamino acids were also conjugated together in a single vaccine construct in an attempt to evaluate the synergy effect of combining multiple adjuvants. Physicochemical characterization demonstrated that the vaccine constructs folded differently and self-assembled into nanoparticles. Significantly, only vaccine constructs containing double copies of lipoamino acids (regardless in conjugation with Q11 or not) were capable to induce significant dendritic cells uptake and subsequent J14-specific antibody responses in non-sizes dependent manners. Q11 had minimal impact in enhancing the immunogenicity of J14 even when it was used in combination with lipoamino acids. These findings highlight the impact of lipoamino acids moiety as a promising immunostimulant carrier and its number of attachment to peptide epitope was found to have a profound effect on the vaccine immunogenicity.
Asunto(s)
Adyuvantes Inmunológicos/química , Lipopéptidos/química , Streptococcus pyogenes/metabolismo , Vacunas Sintéticas/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Dicroismo Circular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Epítopos/inmunología , Femenino , Inmunoglobulina G/análisis , Lipopéptidos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Vacunas Sintéticas/inmunologíaRESUMEN
Vaccine development against group A Streptococcus (GAS) has gained traction in the last decade, fuelled by recognition of the significant worldwide burden of the disease. Several vaccine candidates are currently being evaluated in preclinical and early clinical studies. Here, we investigate two conjugate vaccine candidates that have shown promise in mouse models of infection. Two antigens, the J8 peptide from the conserved C-terminal end of the M protein, and the group A carbohydrate lacking N-acetylglucosamine side chain (ΔGAC) were each conjugated to arginine deiminase (ADI), an anchorless surface protein from GAS. Both conjugate vaccine candidates combined with alum adjuvant were tested in a non-human primate (NHP) model of pharyngeal infection. High antibody titres were detected against J8 and ADI antigens, while high background antibody titres in NHP sera hindered accurate quantification of ΔGAC-specific antibodies. The severity of pharyngitis and tonsillitis signs, as well as the level of GAS colonisation, showed no significant differences in NHPs immunised with either conjugate vaccine candidate compared to NHPs in the negative control group.
RESUMEN
Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.
Asunto(s)
Infecciones Estreptocócicas , Streptococcus , Vacunas , Humanos , Streptococcus pyogenes/genética , Flujo GénicoRESUMEN
A novel vaccine development platform that enables the site-specific conjugation of synthetic lipid adjuvants to recombinant proteins was produced. This technology facilitates the simple and efficient production of homogeneous, chemically-defined, semisynthetic lipoprotein vaccines. Using a polytope 'string-of-beads' approach, a synthetic gene incorporating seven Streptococcus pyogenes M protein strain-specific antigens, and a conserved M protein antigen (J14) was produced, expressed, and attached to a lipoamino acid based adjuvant (lipid core peptide; LCP). Nanoparticles (40 nm diameter) of an optimal size for stimulating antibody-mediated immunity were formed upon the addition of these lipoproteins to aqueous buffer (PBS). Systemic antigen-specific IgG antibodies were raised against all eight antigens in C57BL/6J mice, without the need to formulate with additional adjuvant. These antibodies bound cell surface M proteins of S. pyogenes strains represented within the polytope sequence, with higher antibody levels observed where a dendritic cell targeting peptide (DCpep) was incorporated within the LCP adjuvant. FROM THE CLINICAL EDITOR: In this study, a novel vaccine development system is presented, combining adjuvants with recombinant protein antigens, and presenting the antigen in a nanoparticle system optimized for antibody production. They demonstrate efficient vaccination in a murine model system without the need for additional adjuvants.
Asunto(s)
Adyuvantes Inmunológicos/química , Lípidos/química , Nanopartículas/química , Vacunas Estreptocócicas/inmunología , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Técnica del Anticuerpo Fluorescente , Inmunidad , Lipoproteínas/química , Maleimidas/química , Ratones , Ratones Endogámicos C57BL , Nanopartículas/ultraestructura , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/síntesis química , Vacunas Estreptocócicas/química , Streptococcus pyogenes/inmunologíaRESUMEN
Mutation within the Streptococcus pyogenes (Streptococcus group A; Strep A) covR/S regulatory system has been associated with a hypervirulent phenotype resulting from the upregulation of several virulence factors, including the pore-forming toxin, streptolysin O (SLO). In this study, we utilized a range of covR/S mutants, including M1T1 clonal strains (5448 and a covS mutant generated through mouse passage designated 5448AP), to investigate the contribution of SLO to the pathogenesis of covR/S mutant Strep A disease. Up-regulation of slo in 5448AP resulted in increased SLO-mediated hemolysis, decreased dendritic cell (DC) viability post coculture with Strep A, and increased production of tumor necrosis factor (TNF) and monocyte chemoattractant protein 1 (MCP-1) by DCs. Mouse passage of an isogenic 5448 slo-deletion mutant resulted in recovery of several covR/S mutants within the 5448Δslo background. Passage also introduced mutations in non-covR/S genes, but these were considered to have no impact on virulence. Although slo-deficient mutants exhibited the characteristic covR/S-controlled virulence factor upregulation, these mutants caused increased DC viability with reduced inflammatory cytokine production by infected DCs. In vivo, slo expression correlated with decreased DC numbers in infected murine skin and significant bacteremia by 3 days postinfection, with severe pathology at the infection site. Conversely, the absence of slo in the infecting strain (covR/S mutant or wild-type) resulted in detection of DCs in the skin and attenuated virulence in a murine model of pyoderma. slo-sufficient and -deficient covR/S mutants were susceptible to immune clearance mediated by a combination vaccine consisting of a conserved M protein peptide and a peptide from the CXC chemokine protease SpyCEP. IMPORTANCE Streptococcus pyogenes is responsible for significant numbers of invasive and noninvasive infections which cause significant morbidity and mortality globally. Strep A isolates with mutations in the covR/S system display greater propensity to cause severe invasive diseases, which are responsible for more than 163,000 deaths each year. This is due to the upregulation of virulence factors, including the pore-forming toxin streptolysin O. Utilizing covR/S and slo-knockout mutants, we investigated the role of SLO in virulence. We found that SLO alters interactions with host cell populations and increases Strep A viability at sterile sites of the host, such as the blood, and that its absence results in significantly less virulence. This work underscores the importance of SLO in Strep A virulence while highlighting the complex nature of Strep A pathogenesis. This improved insight into host-pathogen interactions will enable a better understanding of host immune evasion mechanisms and inform streptococcal vaccine development programs.