RESUMEN
Efforts to develop an HIV-1 vaccine include those focusing on conserved structural elements as the target of broadly neutralizing monoclonal antibodies. MAb D5 binds to a highly conserved hydrophobic pocket on the gp41 N-heptad repeat (NHR) coiled coil and neutralizes through prevention of viral fusion and entry. Assessment of 17-mer and 36-mer NHR peptides presenting the D5 epitope in rodent immunogenicity studies showed that the longer peptide elicited higher titers of neutralizing antibodies, suggesting that neutralizing epitopes outside of the D5 pocket may exist. Although the magnitude and breadth of neutralization elicited by NHR-targeting antigens are lower than that observed for antibodies directed to other epitopes on the envelope glycoprotein complex, it has been shown that NHR-directed antibodies are potentiated in TZM-bl cells containing the FcγRI receptor. Herein, we report the design and evaluation of covalently stabilized trimeric 51-mer peptides encompassing the complete gp41 NHR. We demonstrate that these peptide trimers function as effective antiviral entry inhibitors and retain the ability to present the D5 epitope. We further demonstrate in rodent and nonhuman primate immunization studies that our 51-mer constructs elicit a broader repertoire of neutralizing antibody and improved cross-clade neutralization of primary HIV-1 isolates relative to 17-mer and 36-mer NHR peptides in A3R5 and FcγR1-enhanced TZM-bl assays. These results demonstrate that sensitive neutralization assays can be used for structural enhancement of moderately potent neutralizing epitopes. Finally, we present expanded trimeric peptide designs which include unique low-molecular-weight scaffolds that provide versatility in our immunogen presentation strategy.
Asunto(s)
Vacunas contra el SIDA , Anticuerpos Neutralizantes , Anticuerpos Anti-VIH , Proteína gp41 de Envoltorio del VIH , VIH-1 , Proteína gp41 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/química , VIH-1/inmunología , Animales , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Humanos , Ratones , Epítopos/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , Péptidos/inmunología , Péptidos/química , Femenino , Anticuerpos Monoclonales/inmunologíaRESUMEN
mRNA vaccines have recently received significant attention due to their role in combating the SARS-CoV-2 pandemic. As a platform, mRNA vaccines have been shown to elicit strong humoral and cellular immune responses with acceptable safety profiles for prophylactic use. Despite their potential, industrial challenges have limited realization of the vaccine platform on a global scale. Critical among these challenges are supply chain considerations, including mRNA production, cost of goods, and vaccine frozen-chain distribution. Here, we assess the delivery of lipid nanoparticle-encapsulated mRNA (mRNA/LNP) vaccines using a split-dose immunization regimen as an approach to develop mRNA dose-sparing vaccine regimens with potential to mitigate mRNA supply chain challenges. Our data demonstrate that immunization by a mRNA/LNP vaccine encoding respiratory syncytial virus pre-F (RSV pre-F) over a 9 day period elicits comparable or superior magnitude of antibodies when compared to traditional bolus immunization of the vaccine. The split-dose immunization regimens evaluated in our studies were designed to mimic reported drug or antigen release profiles from microneedle patches, highlighting the potential benefit of pairing mRNA vaccines with patch-based delivery technologies to enable sustained release and solid-state stabilization. Overall, our findings provide a proof of concept to support further investigations into the development of sustained delivery approaches for mRNA/LNP vaccines.
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COVID-19 , Infecciones por Virus Sincitial Respiratorio , Vacunas contra Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Humanos , Infecciones por Virus Sincitial Respiratorio/prevención & control , Anticuerpos Antivirales , Vacunas contra Virus Sincitial Respiratorio/genética , SARS-CoV-2/genética , COVID-19/prevención & control , Inmunidad , ARN Mensajero/genética , Anticuerpos NeutralizantesRESUMEN
BACKGROUND: Chlamydia trachomatis is a human pathogen which causes a number of pathologies, including genital tract infections in women that can result in tubal infertility. Prevention of infection and disease control might be achieved through vaccination; however, a safe, efficacious and cost-effective vaccine against C. trachomatis infection remains an unmet medical need. C. trachomatis major outer membrane protein (MOMP), a ß-barrel integral outer membrane protein, is the most abundant antigen in the outer membrane of the bacterium and has been evaluated as a subunit vaccine candidate. Recombinant MOMP (rMOMP) expressed in E. coli cytoplasm forms inclusion bodies and rMOMP extracted from inclusion bodies results in a reduced level of protection compared to the native MOMP in a mouse challenge model. RESULTS: We sought to target the recombinant expression of MOMP to the E. coli outer membrane (OM). Successful surface expression was achieved with codon harmonization, utilization of low copy number vectors and promoters with moderate strength, suitable leader sequences and optimization of cell culture conditions. rMOMP was extracted from E. coli outer membrane, purified, and characterized biophysically. The OM expressed and purified rMOMP is immunogenic in mice and elicits antibodies that react to the native antigen, Chlamydia elementary body (EB). CONCLUSIONS: C. trachomatis MOMP was functionally expressed on the surface of E. coli outer membrane. The OM expressed and purified rMOMP elicits antibodies that react to the native antigen, Chlamydia EB, in a mouse immunogenicity model. Surface expression of MOMP could provide useful reagents for vaccine research, and the methodology could serve as a platform to produce other outer membrane proteins recombinantly.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Chlamydia trachomatis/genética , Escherichia coli/genética , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Vacunas Bacterianas/biosíntesis , Vacunas Bacterianas/química , Células Cultivadas , Infecciones por Chlamydia/prevención & control , Clonación Molecular , ADN Bacteriano/genética , Escherichia coli/metabolismo , Femenino , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/biosíntesis , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Described here is the evaluation of a luciferase (luc) and respiratory syncytial virus (RSV) messenger RNA / lipid nanoparticle (mRNA/LNP) vaccine using a Needle-free Injection System, Tropis®, from PharmaJet® (Golden, Colorado USA). Needle-free jet delivery offers an alternative to needle/syringe. To perform this assessment, compatibility studies with Tropis were first performed with a luc mRNA/LNP and compared to needle/syringe. Although minor changes in particle size and encapsulation efficiency were observed when using Tropis on the benchtop, in vitro luciferase activity remained the same. Next, the luc mRNA/LNP was administered to rats intramuscularly using Tropis or needle/syringe and tracking of the injection and distribution was performed. Lastly, an mRNA encoding a prefusion-stabilized F protein from RSV was delivered intramuscularly using both Tropis and needle/syringe at 1 and 5 mcg mRNA. An equivalent IgG response was observed using both Tropis and needle/syringe. The cell mediated immune (CMI) response was also evaluated, and responses to RSV-F were detected from animals immunized with needle/syringe at all dose levels, and from the animals immunized with Tropis in the 5 and 25 ug groups. These results indicated that delivery of mRNA/LNPs with Tropis is a potential means of administration and an alternative to needle/syringe.
RESUMEN
As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.
Asunto(s)
COVID-19 , Glutamina , Humanos , Glutamina/química , SARS-CoV-2 , Cisteína Endopeptidasas/química , Invenciones , Inhibidores de Proteasas/farmacología , Amidas , Antivirales/farmacología , Antivirales/químicaRESUMEN
Background: MK-1654 is a fully human monoclonal antibody with YTE mutations currently in phase III clinical trials for prophylactic use in protecting infants from human respiratory syncytial virus infection. Materials & methods: We generated anti-idiotype (anti-ID) and anti-YTE antibodies against MK-1654 by panning with MorphoSys HuCal phage libraries, and used the antibodies in the development of MK-1654 pharmacokinetic (PK) and immune response (IR) assays. Results: Detection of MK-1654 in nonhuman primate and human nasal wash samples showed combined use of anti-ID and anti-YTE antibodies can deliver desired sensitivity and accuracy in PK studies. IR studies showed anti-ID can serve as suitable positive control in neutralizing antibody assays. Conclusion: Phage-derived anti-IDs and anti-YTEs are suitable for PK and IR assays.
Asunto(s)
Bacteriófagos , Animales , Humanos , Anticuerpos Neutralizantes , Anticuerpos Monoclonales , InmunidadRESUMEN
Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are related RNA viruses responsible for severe respiratory infections and resulting disease in infants, elderly, and immunocompromised adults1-3. Therapeutic small molecule inhibitors that bind to the RSV polymerase and inhibit viral replication are being developed, but their binding sites and molecular mechanisms of action remain largely unknown4. Here we report a conserved allosteric inhibitory site identified on the L polymerase proteins of RSV and HMPV that can be targeted by a dual-specificity, non-nucleoside inhibitor, termed MRK-1. Cryo-EM structures of the inhibitor in complexes with truncated RSV and full-length HMPV polymerase proteins provide a structural understanding of how MRK-1 is active against both viruses. Functional analyses indicate that MRK-1 inhibits conformational changes necessary for the polymerase to engage in RNA synthesis initiation and to transition into an elongation mode. Competition studies reveal that the MRK-1 binding pocket is distinct from that of a capping inhibitor with an overlapping resistance profile, suggesting that the polymerase conformation bound by MRK-1 may be distinct from that involved in mRNA capping. These findings should facilitate optimization of dual RSV and HMPV replication inhibitors and provide insights into the molecular mechanisms underlying their polymerase activities.
Asunto(s)
Metapneumovirus , Virus Sincitial Respiratorio Humano , Infecciones del Sistema Respiratorio , Lactante , Adulto , Humanos , Anciano , Metapneumovirus/genética , Metapneumovirus/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , ARN MensajeroRESUMEN
Malaria transmission blocking vaccines (TBV) target the mosquito stage of parasite development by passive immunization of mosquitoes feeding on a vaccinated human. Through uptake of vaccine-induced antibodies in a blood meal, mosquito infection is halted and hence transmission to another human host is blocked. Pfs230 is a gametocyte and gamete surface antigen currently under clinical evaluation as a TBV candidate. We have previously shown that chemical conjugation of poorly immunogenic TBV antigens to Exoprotein A (EPA) can enhance their immunogenicity. Here, we assessed Outer Membrane Protein Complex (OMPC), a membrane vesicle derived from Neisseria meningitidis, as a carrier for Pfs230. We prepared Pfs230-OMPC conjugates with varying levels of antigen load and examined immunogenicity in mice. Chemical conjugation of Pfs230 to OMPC enhanced immunogenicity and functional activity of the Pfs230 antigen, and OMPC conjugates achieved 2-fold to 20-fold higher antibody titers than Pfs230-EPA/AdjuPhos® at different doses. OMPC conjugates were highly immunogenic even at low doses, indicating a dose-sparing effect. EPA conjugates induced an IgG subclass profile biased towards a Th2 response, whereas OMPC conjugates induced a strong Th1-biased immune response with high levels of IgG2, which can benefit Pfs230 antibody functional activity, which depends on complement activation. OMPC is a promising carrier for Pfs230 vaccines.
RESUMEN
Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D-K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo-trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR-FTIR) reduction in ß-sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α-helix content increased by 7% (CD) to 13% (ATIR-FTIR). Maintenance of a native-like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Animales , Línea Celular , Chlamydia muridarum/química , Chlamydia trachomatis/química , Cromatografía Líquida de Alta Presión/métodos , Dicroismo Circular/métodos , Cricetulus , Humanos , Peso Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Serogrupo , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Vacunas de Subunidad/químicaRESUMEN
We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC50 4.6 microM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 microM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 microM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr308 and Ser473, reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.
Asunto(s)
Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Péptidos/química , Péptidos/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Homología de Secuencia de Aminoácido , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Adenosina Trifosfato/metabolismo , Proteínas Reguladoras de la Apoptosis , Bencilaminas/farmacología , Unión Competitiva , Proteínas Sanguíneas/inmunología , Carcinoma/química , Carcinoma/metabolismo , Carcinoma/patología , Caspasas/metabolismo , Línea Celular Tumoral , Clonación Molecular , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Compuestos Heterocíclicos con 2 Anillos/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Masculino , Glicoproteínas de Membrana/farmacología , Estructura Molecular , Péptidos/inmunología , Péptidos/metabolismo , Fosfoproteínas/inmunología , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/química , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Quinoxalinas/farmacología , Transducción de Señal/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/farmacología , Neoplasias del Cuello Uterino/química , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
The efficacy of currently licensed anthrax vaccines is largely attributable to a single Bacillus anthracis immunogen, protective antigen. To broaden protection against possible strains resistant to protective antigen-based vaccines, we previously developed a vaccine in which the anthrax polyglutamic acid capsule was covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B and demonstrated that two doses of 2.5µg of this vaccine conferred partial protection of rhesus macaques against inhalational anthrax . Here, we demonstrate complete protection of rhesus macaques against inhalational anthrax with a higher 50µg dose of the same capsule conjugate vaccine. These results indicate that B. anthracis capsule is a highly effective vaccine component that should be considered for incorporation in future generation anthrax vaccines.
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Vacunas contra el Carbunco/inmunología , Carbunco/prevención & control , Cápsulas Bacterianas/inmunología , Ácido Poliglutámico/inmunología , Infecciones del Sistema Respiratorio/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Macaca mulatta , Masculino , Conejos , Vacunas Conjugadas/inmunologíaRESUMEN
The Antigenic Domain 2 (AD-2) is a short region near the N-terminus of glycoprotein B of human cytomegalovirus (HCMV). AD-2 has been shown to contain linear epitopes that are targets for neutralizing monoclonal antibodies from human subjects with natural HCMV infection. However, AD-2 appears to be masked by the adjacent immunodominant AD-1 region. We assessed a serum panel from HCMV-seropositive individuals and found a wide range of antibody titers to AD-2; these did not correlate to serum neutralization. To expose potential epitopes in AD-2, we constructed a series of AD-2 peptide-conjugate vaccines. Mice were immunized 3 times and produced high and sustained antibody titers to AD-2 peptides, but neutralization was weak even after a single boost with whole HCMV virions. Rabbits were likewise immunized with AD-2 peptide vaccines, and produced a robust antibody response, but neutralization was inferior to a recombinant gB vaccine with an oil-in-water adjuvant. These results highlight the challenges of developing a peptide-based vaccine specific to the HCMV gB AD-2 region.
Asunto(s)
Vacunas contra Citomegalovirus/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacunas contra Citomegalovirus/administración & dosificación , Evaluación Preclínica de Medicamentos , Femenino , Ratones Endogámicos BALB C , Conejos , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Proteínas del Envoltorio Viral/administración & dosificaciónRESUMEN
Clostridium difficile is a gram-positive bacterium responsible for a large proportion of nosocomial infections in the developed world. C. difficile secretes toxins A and B (TcdA and TcdB) and both toxins act synergistically to induce a spectrum of pathological responses in infected individuals ranging from pseudomembranous colitis to C. difficile-associated diarrhea. Toxins A and B have been actively investigated as components of prophylactic vaccine as well as targets for therapeutic intervention with antibodies. Expression of such toxins by recombinant technology is often difficult and may require special handling and adherence to strict safety regulations during the manufacturing process due to the inherent toxicity of the proteins. Both toxins are large proteins (308 kDa and 270 kDa, respectively) and contain distinct domains mediating cell attachment, cellular translocation, and enzymatic (glucosidase) activity. Here we describe methods to produce fragments of Toxin B for their subsequent evaluation as components of experimental C. difficile vaccines. Methods presented include selection of fragments encompassing distinct functional regions of Toxin B, purification methods to yield high quality proteins, and analytical evaluation techniques. The approach presented focuses on Toxin B but could be applied to the other component, Toxin A, and/or to any difficult to express or toxic protein.
Asunto(s)
Vacunas Bacterianas/inmunología , Infecciones por Clostridium/prevención & control , Animales , Antígenos Bacterianos/inmunología , Clostridioides difficile/inmunología , Infecciones por Clostridium/inmunología , Diseño de Fármacos , Humanos , Vacunas de Subunidad/inmunologíaRESUMEN
Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F) glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab) is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs) against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Glicoproteínas/inmunología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/uso terapéutico , Virus Sincitiales Respiratorios/inmunología , Virus Sincitiales Respiratorios/patogenicidad , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Técnicas de Visualización de Superficie Celular , Mapeo Epitopo , Epítopos/inmunología , Humanos , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/efectos de los fármacosRESUMEN
Clostridium difficile infection (CDI) is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis, a disease associated with significant morbidity and mortality. The disease is mostly of nosocomial origin, with elderly patients undergoing anti-microbial therapy being particularly at risk. C. difficile produces two large toxins: Toxin A (TcdA) and Toxin B (TcdB). The two toxins act synergistically to damage and impair the colonic epithelium, and are primarily responsible for the pathogenesis associated with CDI. The feasibility of toxin-based vaccination against C. difficile is being vigorously investigated. A vaccine based on formaldehyde-inactivated Toxin A and Toxin B (toxoids) was reported to be safe and immunogenic in healthy volunteers and is now undergoing evaluation in clinical efficacy trials. In order to eliminate cytotoxic effects, a chemical inactivation step must be included in the manufacturing process of this toxin-based vaccine. In addition, the large-scale production of highly toxic antigens could be a challenging and costly process. Vaccines based on non-toxic fragments of genetically engineered versions of the toxins alleviate most of these limitations. We have evaluated a vaccine assembled from two recombinant fragments of TcdB and explored their potential as components of a novel experimental vaccine against CDI. Golden Syrian hamsters vaccinated with recombinant fragments of TcdB combined with full length TcdA (Toxoid A) developed high titer IgG responses and potent neutralizing antibody titers. We also show here that the recombinant vaccine protected animals against lethal challenge with C. difficile spores, with efficacy equivalent to the toxoid vaccine. The development of a two-segment recombinant vaccine could provide several advantages over toxoid TcdA/TcdB such as improvements in manufacturability.
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Proteínas Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Infecciones por Clostridium/prevención & control , Enterocolitis Seudomembranosa/prevención & control , Enterotoxinas/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Neutralizantes/sangre , Clostridioides difficile , Inmunoglobulina G/sangre , Masculino , Mesocricetus , Pruebas de Neutralización , Proteínas Recombinantes/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
Antibodies that neutralize infectivity of malaria sporozoites target the central repeat region of the circumsporozoite (CS) protein, which in Plasmodium falciparum is comprised primarily of 30-40 tandem NANP tetramer repeats. We evaluated immunogenicity of an alum-adsorbed (NANP)(6) peptide conjugated to an outer membrane protein complex (OMPC) derived from Neisseria meningitidis, a carrier protein used in a licensed Haemophilus influenzae pediatric vaccine. Mice immunized with (NANP)(6)-OMPC adsorbed to Merck's alum adjuvant (MAA), with or without Iscomatrix® as co-adjuvant, developed high levels of anti-repeat peptide antibody that inhibited in vitro invasion of human hepatoma cells by transgenic P. berghei sporozoites that express P. falciparum CS repeats (PfPb). Inhibition of sporozoite invasion in vitro correlated with in vivo resistance to challenge by the bites of PfPb-infected mosquitoes. Challenged mice had >90% reduction of hepatic stage parasites as measured by real-time PCR, and either sterile immunity, i.e., no detectable blood stage parasites, or delayed prepatent periods which indicate neutralization of a majority, but not all, sporozoites. Rhesus macaques immunized with two doses of (NANP)(6)-OMPC/MAA formulated with Iscomatrix® developed anti-repeat antibodies that persisted for ~2 years. A third dose of (NANP)(6)-OMPC/MAA+ Iscomatrix® at that time elicited strong anamnestic antibody responses. Rhesus macaque immune sera obtained post second and third dose of vaccine displayed high levels of sporozoite neutralizing activity in vitro that correlated with presence of high anti-repeat antibody titers. These preclinical studies in mice of different MHC haplotypes and a non-human primate support use of CS peptide-OMPC conjugates as a highly immunogenic platform to evaluate CS protective epitopes. Potential pre-erythrocytic vaccines can be combined with sexual blood stage vaccines as a multi-antigen malaria vaccine to block invasion and transmission of Plasmodium parasites.