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1.
Proc Natl Acad Sci U S A ; 121(22): e2317230121, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38768344

ABSTRACT

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.


Subject(s)
AIDS Vaccines , Antibodies, Neutralizing , HIV Antibodies , HIV Envelope Protein gp41 , HIV-1 , HIV Envelope Protein gp41/immunology , HIV Envelope Protein gp41/chemistry , HIV-1/immunology , Animals , AIDS Vaccines/immunology , Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , Humans , Mice , Epitopes/immunology , HIV Infections/immunology , HIV Infections/prevention & control , HIV Infections/virology , Peptides/immunology , Peptides/chemistry , Female , Antibodies, Monoclonal/immunology
2.
Open Forum Infect Dis ; 11(5): ofae220, 2024 May.
Article in English | MEDLINE | ID: mdl-38770212

ABSTRACT

Global use of pneumococcal conjugate vaccines (PCVs) with increasingly broader serotype coverage has helped to reduce the burden of pneumococcal disease in children and adults. In clinical studies comparing PCVs, higher-valency PCVs have met noninferiority criteria (based on immunoglobulin G geometric mean concentrations and response rates) for most shared serotypes. A numeric trend of declining immunogenicity against shared serotypes with higher-valency PCVs has also been observed; however, the clinical relevance is uncertain, warranting additional research to evaluate the effectiveness of new vaccines. Novel conjugation processes, carriers, adjuvants, and vaccine platforms are approaches that could help maintain or improve immunogenicity and subsequent vaccine effectiveness while achieving broader protection with increasing valency in pneumococcal vaccines.

3.
J Chromatogr A ; 1705: 464194, 2023 Aug 30.
Article in English | MEDLINE | ID: mdl-37419021

ABSTRACT

Continuous multi-column chromatography (CMCC) has been successfully implemented to address biopharmaceutical biomolecule instability, to improve process efficiency, and to reduce facility footprint and capital cost. This paper explores the implementation of a continuous multi-membrane chromatography (CMMC) process, using four membrane units, for a large viral particle in just few weeks. CMMC improves the efficiency of the chromatography step by enabling higher loads with smaller membranes for multiple cycles of column use and enables steady-state continuous bioprocessing. The separation performance of CMMC was compared to a conventional batch chromatographic capture step used at full manufacturing scale. The product step yield was 80% using CMMC versus 65% in batch mode while increasing slightly the relative purity. Furthermore, the total amount of membrane area required for the CMMC approach was approximately 10% of the area needed for batch operation, while realizing similar processing times. Since CMMC uses smaller membrane sizes, it can take advantage of the high flow rates achievable for membrane chromatography that are not typically possible at larger membrane scales due to skid flow rate limitations. As such, CMMC offers the potential for more efficient and cost-effective purification trains.


Subject(s)
Antibodies, Monoclonal , Biological Products , Chromatography , Staphylococcal Protein A/chemistry
4.
Vaccine ; 41(4): 903-913, 2023 Jan 23.
Article in English | MEDLINE | ID: mdl-36566163

ABSTRACT

Despite the widespread effectiveness of pneumococcal conjugate vaccines on the overall incidence of invasive pneumococcal disease, the global epidemiological landscape continues to be transformed by residual disease from non-vaccine serotypes, thus highlighting the need for vaccines with expanded disease coverage. To address these needs, we have developed V116,an investigational 21-valent non-adjuvanted pneumococcal conjugate vaccine (PCV),containingpneumococcal polysaccharides (PnPs) 3, 6A, 7F, 8, 9N, 10A, 11A,12F, 15A, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F, 35B, anda de-O-acetylated 15B(deOAc15B) individually conjugated to the nontoxic diphtheria toxoid CRM197 carrier protein. Preclinical studies evaluated the immunogenicity of V116 inadult monkeys, rabbits, and mice. Following one dose, V116 was found to be immunogenic in preclinical animal species and induced functional antibodies for all serotypes included in the vaccine, in addition to cross-reactive functional antibodies to serotypes 6C and 15B. In these preclinical animal studies, the increased valency of V116 did not result in serotype-specific antibody suppression when compared to lower valent vaccines V114 or PCV13. In addition, when compared with naïve controls, splenocytes from V116 to immunized animals demonstrated significant induction of CRM197-specific T cells in both IFN-γ and IL-4 ELISPOT assays, as well as Th1 and Th2 cytokine induction through in vitro stimulation assays, thus suggesting the ability of V116 to engage T cell dependent immune response pathways to aid in development of memory B cells. V116 also demonstrated significant protection in mice from intratracheal challenge with serotype 24F, a novel serotype not contained in any currently licensed vaccine.


Subject(s)
Pneumococcal Infections , Streptococcus pneumoniae , Rabbits , Mice , Animals , Pneumococcal Vaccines , Vaccines, Conjugate , Macaca mulatta , Antibodies, Bacterial , Pneumococcal Infections/prevention & control , Serogroup , Disease Models, Animal
5.
Lancet Infect Dis ; 23(2): 233-246, 2023 02.
Article in English | MEDLINE | ID: mdl-36116461

ABSTRACT

BACKGROUND: A pneumococcal conjugate vaccine (PCV) specifically focused on serotypes associated with adult residual disease burden is urgently needed. We aimed to assess V116, an investigational 21-valent PCV, that contains pneumococcal polysaccharides (PnPs), which account for 74-94% of invasive pneumococcal disease in adults aged 65 years or older. METHODS: We did a phase 1/2, randomised, double-blind, active comparator-controlled, multicentre, non-inferiority and superiority trial. The phase 1 study was done at two clinical sites in the USA, and the phase 2 study was done in 18 clinical sites in the USA. Eligible participants were healthy adults with or without chronic medical conditions assessed as stable, aged 18-49 years in the phase 1 trial and aged 50 years or older in the phase 2 trial. Participants were excluded if they had a history of invasive pneumococcal disease or other culture-positive pneumococcal disease within the past 3 years, known hypersensitivity to a vaccine component, known or suspected impairment of immunological function, were pregnant or were breastfeeding, or had previously received any pneumococcal vaccine. Participants had to abstain from sexual activity or use protocol approved contraception. All participants were centrally randomly assigned to a vaccine group using an interactive response technology system. Participants and investigators were masked to group assignment. In phase 1, participants were randomly assigned (1:1:1) to receive a single dose of V116-1 (2 µg per pneumococcal polysaccharide [PnP] per 0·5 mL) or V116-2 (4 µg per PnP per 1·0 mL) or the 23-valent unconjugated PnP vaccine, PPSV23 (25 µg per PnP per 0·5 mL). In phase 2, participants were randomly assigned (1:1) to receive one dose of V116 (4 µg per PnP per 1·0 mL) or PPSV23 (25 µg per PnP per 0·5 mL), stratified by age. Safety analyses included all randomly assigned participants who received study vaccine; immunogenicity analyses were per protocol. For both phases, the primary safety outcome was the proportion of participants with solicited injection-site adverse events and solicited systemic adverse events up to day 5 after vaccination and the proportion of participants with vaccine-related serious adverse events to 6 months after vaccination. In phase 2, primary immunogenicity outcomes were to test non-inferiority of V116 compared with PPSV23 as measured by serotype-specific opsonophagocytic antibody geometric mean titres (OPA-GMT) ratios for the serotypes common to the two vaccines at 30 days after vaccination (using a 0·33 margin) and to test superiority of V116 compared with PPSV23 as measured by serotype-specific OPA-GMT ratios for the serotypes unique to V116 at 30 days after vaccination (using a 1·0 margin). This trial is registered with Clinicaltrials.gov, NCT04168190. FINDINGS: Between Dec 6 and 26, 2019, 92 volunteers were screened and 90 (98%) enrolled for phase 1 (59 [66%] women; 31 [34%] men); 30 participants were assigned to each group and received study vaccine. 30 (100%) participants in the V116-1 group, 29 (97%) in the V116-2 group, and 30 (100%) participants in the PPSV23 group were included in the per-protocol immunogenicity evaluation. From Sept 23, 2020, to Jan 12, 2021, 527 volunteers were screened, and 510 (97%) participants were enrolled in the phase 2 trial. 508 participants (>99%; 254 [100%] of 254 participants randomly assigned to the V116 group and 254 [99%] of 256 randomly assigned to PPSV23 group) received study vaccine (281 [55%] women; 227 [45%] men). 252 (99%) of 254 of participants in the V116 group and 254 (99%) of 256 participants in the PPSV23 group were included in the primary immunogenicity analyses. There were no vaccine-related serious adverse events or vaccine-related deaths in either study phase. In both phases, the most common solicited injection site adverse event was injection site pain (phase 1 22 [73%] participants in V116-1 group, 23 [77%] participants in V116-2 group, and 17 [57%] participants in the PPSV23 group; phase 2 118 [46%] of 254 participants in the V116 group and 96 [38%] of 254 in the PPSV23 group]. The most common solicited systemic adverse events in phase 1 was fatigue (eight [27%] participants in the V116-1 group, eight [27%] participants in the V116-2 group, and five [17%] participants in PPSV23 group) and myalgia (eight [27%] participants in the V116-1 group, nine (30%) participants in the V116-2 group, and four (13%) participants in the PPSV23 group]. In phase 2, the most frequently reported solicited systemic adverse event was fatigue (49 [19%] participants in V116 group, and 31 [12%] participants in PPSV23 group). In both phases, most of the solicited adverse events in all vaccine groups were mild and of short duration (≤3 days). V116 met non-inferiority criteria compared with PPSV23 for the 12 shared serotypes and met superiority criteria compared to PPSV23 for the nine unique serotypes. INTERPRETATION: V116 was well tolerated with a safety profile generally similar to PPSV23; consistent with licensed pneumococcal conjugate vaccines. Functional OPA antibodies were induced to all V116 vaccine serotypes. The vaccine was non-inferior to PPSV23 for the 12 serotypes common to both vaccines and superior to PPSV23 for the nine unique serotypes in V116. Our findings support the development of V116 for prevention of pneumococcal disease in adults. FUNDING: Merck Sharp & Dohme, subsidiary of Merck & Co, Rahway, NJ, USA.


Subject(s)
Pneumococcal Infections , Streptococcus pneumoniae , Male , Humans , Adult , Female , Vaccines, Conjugate , Vaccination/methods , Pneumococcal Vaccines , Pneumococcal Infections/prevention & control , Pneumococcal Infections/drug therapy , Double-Blind Method , Injection Site Reaction , Immunogenicity, Vaccine
6.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Article in English | MEDLINE | ID: mdl-33431684

ABSTRACT

The HIV-1 gp41 N-heptad repeat (NHR) region of the prehairpin intermediate, which is transiently exposed during HIV-1 viral membrane fusion, is a validated clinical target in humans and is inhibited by the Food and Drug Administration (FDA)-approved drug enfuvirtide. However, vaccine candidates targeting the NHR have yielded only modest neutralization activities in animals; this inhibition has been largely restricted to tier-1 viruses, which are most sensitive to neutralization by sera from HIV-1-infected individuals. Here, we show that the neutralization activity of the well-characterized NHR-targeting antibody D5 is potentiated >5,000-fold in TZM-bl cells expressing FcγRI compared with those without, resulting in neutralization of many tier-2 viruses (which are less susceptible to neutralization by sera from HIV-1-infected individuals and are the target of current antibody-based vaccine efforts). Further, antisera from guinea pigs immunized with the NHR-based vaccine candidate (ccIZN36)3 neutralized tier-2 viruses from multiple clades in an FcγRI-dependent manner. As FcγRI is expressed on macrophages and dendritic cells, which are present at mucosal surfaces and are implicated in the early establishment of HIV-1 infection following sexual transmission, these results may be important in the development of a prophylactic HIV-1 vaccine.


Subject(s)
HIV Envelope Protein gp41/immunology , HIV Infections/drug therapy , Receptors, IgG/immunology , Repetitive Sequences, Amino Acid/genetics , Amino Acid Sequence , Animals , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibody Affinity , Guinea Pigs , HIV Antibodies/immunology , HIV Antibodies/pharmacology , HIV Envelope Protein gp41/genetics , HIV Infections/genetics , HIV Infections/immunology , HIV Infections/virology , HIV Seropositivity/immunology , HIV-1/drug effects , HIV-1/immunology , HIV-1/pathogenicity , Humans , Immune Sera/immunology , Immune Sera/pharmacology , Immunization , Immunoglobulin G/immunology , Repetitive Sequences, Amino Acid/immunology , Virus Internalization/drug effects
8.
Front Immunol ; 9: 2194, 2018.
Article in English | MEDLINE | ID: mdl-30319643

ABSTRACT

The exploitation of various human immunodeficiency virus type-1 (HIV-1) vaccines has posed great challenges for the researchers in precisely evaluating the vaccine-induced immune responses, however, the understanding of vaccination response suffers from the lack of unbiased characterization of the immune landscape. The rapid development of high throughput sequencing (HTS) makes it possible to scrutinize the extremely complicated immunological responses during vaccination. In the current study, three vaccines, namely N36, N51, and 5-Helix based on the HIV-1 gp41 pre-hairpin fusion intermediate were applied in rhesus macaques. We assessed the longitudinal vaccine responses using HTS, which delineated the evolutionary features of both T cell and B cell receptor repertoires with extreme diversities. Upon vaccination, we unexpectedly found significant discrepancies in the landscapes of T-cell and B-cell repertoires, together with the detection of significant class switching and the lineage expansion of the B cell receptor or immunoglobulin heavy chain (IGH) repertoire. The vaccine-induced expansions of lineages were further evaluated for mutation rate, lineage abundance, and lineage size features in their IGH repertoires. Collectively, these findings conclude that the N51 vaccine displayed superior performance in inducing the class-switch of B cell isotypes and promoting mutations of IgM B cells. In addition, the systematic HTS analysis of the immune repertoires demonstrates its wide applicability in enhancing the understanding of immunologic changes during pathogen challenge, and will guide the development, evaluation, and exploitation of new generation of diagnostic markers, immunotherapies, and vaccine strategies.


Subject(s)
AIDS Vaccines/immunology , B-Lymphocytes/immunology , HIV Envelope Protein gp41/immunology , HIV-1/immunology , T-Lymphocytes/immunology , AIDS Vaccines/administration & dosage , Animals , B-Lymphocytes/metabolism , Disease Models, Animal , Female , High-Throughput Nucleotide Sequencing , Humans , Immunogenicity, Vaccine , Immunoglobulin M/genetics , Immunoglobulin M/immunology , Macaca mulatta , Male , Mutation , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/immunology , T-Lymphocytes/metabolism
9.
Protein Sci ; 27(11): 1923-1941, 2018 11.
Article in English | MEDLINE | ID: mdl-30144190

ABSTRACT

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.


Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Animals , Cell Line , Chlamydia muridarum/chemistry , Chlamydia trachomatis/chemistry , Chromatography, High Pressure Liquid/methods , Circular Dichroism/methods , Cricetulus , Humans , Molecular Weight , Protein Structure, Quaternary , Protein Structure, Tertiary , Serogroup , Spectroscopy, Fourier Transform Infrared/methods , Vaccines, Subunit/chemistry
10.
AANA J ; 85(1): 61-71, 2017 Feb.
Article in English | MEDLINE | ID: mdl-31554560

ABSTRACT

Successful intubation of the patient with a difficult airway marks the beginning of the challenges facing the nurse anesthetist charged with delivery of that patient's anesthesia care. Too often, on successful intubation of this patient, the anesthetist and other members of the perioperative team may relax too much. Substantial planning and consultations have been employed to achieve the successful intubation of the patient with the difficult airway. Yet frequently, the final aspect of any general anesthetic-extubation-does not receive sufficient planning even though there continues to be a large volume of critical airway incidents surrounding the extubation and transportation of such a patient to the postanesthesia care unit.

11.
J Biol Chem ; 292(1): 278-291, 2017 Jan 06.
Article in English | MEDLINE | ID: mdl-27879316

ABSTRACT

A major goal for HIV-1 vaccine development is an ability to elicit strong and durable broadly neutralizing antibody (bNAb) responses. The trimeric envelope glycoprotein (Env) spikes on HIV-1 are known to contain multiple epitopes that are susceptible to bNAbs isolated from infected individuals. Nonetheless, all trimeric and monomeric Env immunogens designed to date have failed to elicit such antibodies. We report the structure-guided design of HIV-1 cyclically permuted gp120 that forms homogeneous, stable trimers, and displays enhanced binding to multiple bNAbs, including VRC01, VRC03, VRC-PG04, PGT128, and the quaternary epitope-specific bNAbs PGT145 and PGDM1400. Constructs that were cyclically permuted in the V1 loop region and contained an N-terminal trimerization domain to stabilize V1V2-mediated quaternary interactions, showed the highest homogeneity and the best antigenic characteristics. In guinea pigs, a DNA prime-protein boost regimen with these new gp120 trimer immunogens elicited potent neutralizing antibody responses against highly sensitive Tier 1A isolates and weaker neutralizing antibody responses with an average titer of about 115 against a panel of heterologous Tier 2 isolates. A modest fraction of the Tier 2 virus neutralizing activity appeared to target the CD4 binding site on gp120. These results suggest that cyclically permuted HIV-1 gp120 trimers represent a viable platform in which further modifications may be made to eventually achieve protective bNAb responses.


Subject(s)
Antibodies, Neutralizing/blood , Drug Design , HIV Antibodies/blood , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp120/metabolism , HIV Infections/immunology , HIV-1/immunology , Animals , Antibodies, Neutralizing/immunology , Binding Sites , Crystallography, X-Ray , Epitopes/immunology , Guinea Pigs , HIV Antibodies/immunology , HIV Infections/blood , HIV Infections/virology , Humans , Protein Binding , Protein Conformation , Protein Multimerization
12.
Vaccine ; 34(34): 4012-6, 2016 07 25.
Article in English | MEDLINE | ID: mdl-27329184

ABSTRACT

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.


Subject(s)
Anthrax Vaccines/immunology , Anthrax/prevention & control , Bacterial Capsules/immunology , Polyglutamic Acid/immunology , Respiratory Tract Infections/prevention & control , Animals , Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Bacterial Toxins/immunology , Female , Immunoglobulin G/blood , Immunoglobulin M/blood , Macaca mulatta , Male , Rabbits , Vaccines, Conjugate/immunology
13.
Methods Mol Biol ; 1403: 385-96, 2016.
Article in English | MEDLINE | ID: mdl-27076142

ABSTRACT

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.


Subject(s)
Bacterial Vaccines/immunology , Clostridium Infections/prevention & control , Animals , Antigens, Bacterial/immunology , Clostridioides difficile/immunology , Clostridium Infections/immunology , Drug Design , Humans , Vaccines, Subunit/immunology
14.
MAbs ; 7(4): 707-18, 2015.
Article in English | MEDLINE | ID: mdl-25996084

ABSTRACT

Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.


Subject(s)
Antibodies, Monoclonal , Antibodies, Viral , Antibody-Producing Cells/immunology , Dengue Vaccines/immunology , Dengue Virus/immunology , Immunoglobulin Variable Region , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Macaca mulatta , Sequence Analysis, Protein
15.
Vaccine ; 32(24): 2812-8, 2014 May 19.
Article in English | MEDLINE | ID: mdl-24662701

ABSTRACT

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.


Subject(s)
Bacterial Proteins/immunology , Bacterial Toxins/immunology , Bacterial Vaccines/immunology , Clostridium Infections/prevention & control , Enterocolitis, Pseudomembranous/prevention & control , Enterotoxins/immunology , Animals , Antibodies, Bacterial/blood , Antibodies, Neutralizing/blood , Clostridioides difficile , Immunoglobulin G/blood , Male , Mesocricetus , Neutralization Tests , Recombinant Proteins/immunology , Vaccines, Synthetic/immunology
16.
Telemed J E Health ; 20(1): 94-6, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24160899

ABSTRACT

The use of Facebook is ubiquitous among both patients and physicians. Often Facebook intrudes into medical practice, thereby highlighting its potential to be either a positive or negative factor in a patient's medical care. Despite being a "hot topic" in the medical literature, very few real world examples exist of physicians actually using information obtained from Facebook to reach a diagnosis or otherwise affect patient care. We present a case involving a 13-year-old girl who posted photographs and captions on Facebook demonstrating suicidal ideation. The patient's parents were alerted to the girl's statements in her Facebook profile and brought her to the emergency department. The girl's statements and photographs, as reported by her parents, were used by an emergency physician to make a diagnosis of suicidal risk and to disposition of the patient to an inpatient psychiatric ward. We discuss the potential diagnostic utility of information posted on Facebook and briefly discuss the ethical questions surrounding this situation.


Subject(s)
Emergency Service, Hospital , Social Networking , Suicidal Ideation , Adolescent , Female , Humans
17.
Hum Vaccin Immunother ; 9(3): 488-96, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23249976

ABSTRACT

Vaccine development for Group A streptococcal (GAS) infection has been extensively focused on the N-terminal hypervariable or the C-terminal conserved regions of the M protein, a major virulence factor of GAS. We evaluated the immunogenicity and functional activity of the conserved C-terminal peptide vaccine candidate, J8, conjugated to CRM197, in two mouse strains: C3H (H2(k)) and Balb/c (H2(d)), and in rhesus macaques. Mice were immunized with J8-CRM197 formulated with Amorphous Aluminum Hydroxyphosphate Sulfate Adjuvant (AAHSA), and non-human primates were immunized with J8-CRM197 formulated with AAHSA, ISCOMATRIX (TM) adjuvant, or AAHSA/ISCOMATRIX adjuvant. J8-CRM197 was immunogenic in mice from both H2(k) and H2(d) backgrounds, and the antibodies generated bound to the surface of four different GAS serotypes and had functional bacterial opsonic activity. Mice immunized with J8-CRM197/AAHSA demonstrated varying degrees of protection from lethal challenge. We also demonstrated that J8-CRM197 is immunogenic in non-human primates. Our data confirm the utility of J8 as a potential GAS vaccine candidate and demonstrate that CRM197 is an acceptable protein carrier for this peptide.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Bacterial Proteins/administration & dosage , Streptococcal Infections/prevention & control , Streptococcal Vaccines/immunology , Streptococcus pyogenes/immunology , Vaccines, Subunit/immunology , Adjuvants, Immunologic/metabolism , Animals , Bacterial Proteins/metabolism , Disease Models, Animal , Female , Macaca mulatta , Mice, Inbred BALB C , Mice, Inbred C3H , Streptococcal Infections/immunology , Streptococcal Vaccines/administration & dosage , Streptococcal Vaccines/genetics , Streptococcal Vaccines/metabolism , Streptococcus pyogenes/genetics , Survival Analysis , Vaccines, Conjugate/administration & dosage , Vaccines, Conjugate/genetics , Vaccines, Conjugate/immunology , Vaccines, Conjugate/metabolism , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/genetics , Vaccines, Subunit/metabolism , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vaccines, Synthetic/metabolism
18.
Vaccine ; 30(5): 846-52, 2012 Jan 20.
Article in English | MEDLINE | ID: mdl-22172509

ABSTRACT

Bacillus anthracis, the causative agent of anthrax, is recognized as one of the most serious bioterrorism threats. The current human vaccines are based on the protective antigen component of the anthrax toxins. Concern about possible vaccine resistant strains and reliance on a single antigen has prompted the search for additional immunogens. Bacterial capsules, as surface-expressed virulence factors, are well-established components of several licensed vaccines. In a previous study we showed that an anthrax vaccine consisting of the B. anthracis poly-γ-D-glutamic acid capsule covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B protected mice against parenteral B. anthracis challenge. Here we tested this vaccine in rabbits and monkeys against an aerosol spore challenge. The vaccine induced anti-capsule antibody responses in both species, measured by ELISA and a macrophage opsono-adherence assay. While rabbits were not protected against a high aerosol challenge dose, significant protection was observed in monkeys receiving the capsule conjugate vaccine. The results confirm that the capsule is a protective immunogen against anthrax, being the first non-toxin antigen shown to be efficacious in monkeys and suggest that addition of capsule may broaden and enhance the protection afforded by protective antigen-based vaccines.


Subject(s)
Anthrax Vaccines/immunology , Anthrax/prevention & control , Bacterial Capsules/immunology , Bacterial Outer Membrane Proteins/chemistry , Animals , Anthrax/immunology , Anthrax Vaccines/administration & dosage , Antibodies, Bacterial/blood , Bacterial Capsules/chemistry , Bacterial Outer Membrane Proteins/isolation & purification , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Female , Macaca mulatta , Macrophages/immunology , Male , Neisseria meningitidis/chemistry , Opsonin Proteins/blood , Phagocytosis , Rabbits , Survival Analysis , Vaccines, Conjugate/administration & dosage , Vaccines, Conjugate/immunology
19.
J Biol Chem ; 285(52): 40604-11, 2010 Dec 24.
Article in English | MEDLINE | ID: mdl-20943652

ABSTRACT

We describe here a novel platform technology for the discovery of small molecule mimetics of conformational epitopes on protein antigens. As a model system, we selected mimetics of a conserved hydrophobic pocket within the N-heptad repeat region of the HIV-1 envelope protein, gp41. The human monoclonal antibody, D5, binds to this target and exhibits broadly neutralizing activity against HIV-1. We exploited the antigen-binding property of D5 to select complementary small molecules using a high throughput screen of a diverse chemical collection. The resulting small molecule leads were rendered immunogenic by linking them to a carrier protein and were shown to elicit N-heptad repeat-binding antibodies in a fraction of immunized mice. Plasma from HIV-1-infected subjects shown previously to contain broadly neutralizing antibodies was found to contain antibodies capable of binding to haptens represented in the benzylpiperidine leads identified as a result of the high throughput screen, further validating these molecules as vaccine leads. Our results suggest a new paradigm for vaccine discovery using a medicinal chemistry approach to identify lead molecules that, when optimized, could become vaccine candidates for infectious diseases that have been refractory to conventional vaccine development.


Subject(s)
AIDS Vaccines/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , HIV Envelope Protein gp41/immunology , HIV Infections/immunology , HIV-1/immunology , Peptidomimetics/immunology , AIDS Vaccines/pharmacology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Female , HIV Infections/blood , HIV Infections/prevention & control , Haptens/immunology , Haptens/pharmacology , Humans , Mice , Mice, Inbred BALB C , Peptidomimetics/pharmacology
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