Catherine A. Cosgrove

Characterization of Salmonella enterica Derivatives Harboring Defined aroC and Salmonella Pathogenicity Island 2 Type III Secretion System (ssaV) Mutations by Immunization of Healthy Volunteers

ABSTRACT The attenuation and immunogenicity of two novel Salmonella vaccine strains, Salmonella enterica serovar Typhi (Ty2 ΔaroC ΔssaV, designated ZH9) and S. enterica serovar Typhimurium (TML ΔaroC ΔssaV, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 107, 108, or 109 CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 108 and two of three receiving 109 CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 108 and 109 CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 109 CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi.

Protective Levels of Diphtheria-Neutralizing Antibody Induced in Healthy Volunteers by Unilateral Priming-Boosting Intranasal Immunization Associated with Restricted Ipsilateral Mucosal Secretory Immunoglobulin A

ABSTRACT Subunit intranasal vaccines offer the prospect of inducing combined systemic-mucosal immunity against mucosally transmitted infections such as human immunodeficiency virus. However, although human studies have demonstrated the induction of active immunity, secretory immunoglobulin A (sIgA) responses are variable, and no study has demonstrated protection by accepted vaccine-licensing criteria as measured by direct toxin-neutralizing activity. Using the genetically inactivated mutant diphtheria toxoid CRM197 in a bioadhesive polycationic polysaccharide chitosan delivery system, we found that a single nasal immunization was well tolerated and boosted antitoxin neutralizing activity in healthy volunteers, which could be further boosted by a second immunization. The neutralizing activity far exceeded accepted protective levels and was equivalent to that induced by standard intramuscular vaccine and significantly greater than intranasal immunization with CRM197 in the absence of chitosan. A striking but unexpected observation was that although unilateral intranasal immunization induced circulating antitoxin antibody-secreting cells, a nasal antitoxin sIgA response was seen only after the second immunization and only in the vaccinated nostril. If these data are reproduced in larger studies, an intranasal diphtheria vaccine based on CRM197-chitosan could be rapidly licensed for human use. However, a restricted sIgA response suggests that care must be taken in the priming-boosting strategy and clinical sampling techniques when evaluating such vaccines for the induction of local mucosal immunity.

Clinical Application of Whole-Genome Sequencing To Inform Treatment for Multidrug-Resistant Tuberculosis Cases

ABSTRACT The treatment of drug-resistant tuberculosis cases is challenging, as drug options are limited, and the existing diagnostics are inadequate. Whole-genome sequencing (WGS) has been used in a clinical setting to investigate six cases of suspected extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) encountered at a London teaching hospital between 2008 and 2014. Sixteen isolates from six suspected XDR-TB cases were sequenced; five cases were analyzed in a clinically relevant time frame, with one case sequenced retrospectively. WGS identified mutations in the M. tuberculosis genes associated with antibiotic resistance that are likely to be responsible for the phenotypic resistance. Thus, an evidence base was developed to inform the clinical decisions made around antibiotic treatment over prolonged periods. All strains in this study belonged to the East Asian (Beijing) lineage, and the strain relatedness was consistent with the expectations from the case histories, confirming one contact transmission event. We demonstrate that WGS data can be produced in a clinically relevant time scale some weeks before drug sensitivity testing (DST) data are available, and they actively help clinical decision-making through the assessment of whether an isolate (i) has a particular resistance mutation where there are absent or contradictory DST results, (ii) has no further resistance markers and therefore is unlikely to be XDR, or (iii) is identical to an isolate of known resistance (i.e., a likely transmission event). A small number of discrepancies between the genotypic predictions and phenotypic DST results are discussed in the wider context of the interpretation and reporting of WGS results.

Induction of Protective Serum Meningococcal Bactericidal and Diphtheria-Neutralizing Antibodies and Mucosal Immunoglobulin A in Volunteers by Nasal Insufflations of the Neisseria meningitidis Serogroup C Polysaccharide-CRM197 Conjugate Vaccine Mixed with Chitosan

ABSTRACT Thirty-six healthy volunteers received either a single intramuscular injection of Neisseria meningitidis serogroup C polysaccharide (MCP)-CRM197 conjugate vaccine in alum or two nasal insufflations 28 days apart of the same vaccine powder, without alum, mixed with chitosan. Nasal immunization was well tolerated, with fewer symptoms reported than after intramuscular injection. The geometric mean concentrations of MCP-specific immunoglobulin G (IgG) after one nasal immunization were 3.25 μg/ml in naïve subjects and 14.4 μg/ml in subjects previously immunized parenterally, compared with 4.30 μg/ml in naïve subjects immunized intramuscularly. The geometric mean titer of serum bactericidal antibody (SBA) rose 24-fold after two nasal immunizations in naïve subjects and was comparable to parenteral immunization (1,080 versus 1,625). All subjects achieved SBA titers associated with protection after two nasal immunizations: even those with titers of <8 at entry. A single nasal immunization boosted the SBA titer to ≥128 in 96% of previously immunized subjects, and two immunizations achieved this level in 92% of naive subjects. MCP-specific IgG levels were ∼70% IgG2 and ∼20% IgG1 after nasal or intramuscular immunization. Increases in CRM197-specific IgG and diphtheria toxin-neutralizing activity were observed after nasal or intramuscular immunization, with balanced IgG1/IgG2 and higher IgG4. Significant MCP-specific secretory IgA was detected in nasal wash only after nasal immunization and predominantly on the immunized side. Simple nasal insufflation of existing MCP-CRM197 conjugate vaccines in chitosan offers an inexpensive but effective needle-free prime and boost against serogroup C N. meningitidis and diphtheria.

Boosting of Cellular Immunity against Mycobacterium tuberculosis and Modulation of Skin Cytokine Responses in Healthy Human Volunteers by Mycobacterium bovis BCG Substrain Moreau Rio de Janeiro Oral Vaccine

ABSTRACT Oral immunization of healthy adults with 107 CFU BCG Moreau Rio de Janeiro was well tolerated and significantly boosted gamma interferon responses to purified protein derivative, Ag85, and MPB70 from previous childhood intradermal BCG immunization. Oral BCG offers the possibility of a needle-free tuberculosis vaccine and of boosting the protective immunity from intradermal tuberculosis vaccines.

Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV

Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb.

XDR-TB transmission in London: Case management and contact tracing investigation assisted by early whole genome sequencing

OBJECTIVES We describe the first published cluster of extensively drug resistant Tuberculosis (XDR-TB) in the UK and show how early whole genome sequencing (WGS) of Mtb can assist in case management and contact investigations. METHODS We describe the contact tracing investigation undertaken after the presentation of an adult with XDR-TB. Active cases were treated with an XDR-TB drug regimen and contacts underwent a programme of follow-up for 2 years. All isolates of Mycobacterium tuberculosis (Mtb) were assessed early using whole genome sequencing (WGS) as well as routine drug susceptibility testing (DST). RESULTS Thirty-three contacts were screened. In the first year one confirmed and one probable case were identified through contact tracing. A further possible case was identified through epidemiological links. Two confirmed cases were identified through WGS 2 years later. Twenty-five (80%) contacts without evidence of tuberculosis were adherent to 1 year of follow-up and 14 (45%) were adherent to 2 years of follow-up. WGS of Mtb was used to guide drug choices, rapidly identify transmission events, and alter public health management. CONCLUSION WGS of Mtb enabled rapid effective individualized treatment and facilitated public health interventions by early identification of transmission events.

Phase 1 clinical trial of live attenuated Shigella dysenteriae type-1 ΔicsA Δent Δfep ΔstxA: HgR oral vaccine SC599 in healthy human adult volunteers

Twenty-eight adults received between 10(2) and 10(8)colony forming units of live Shigella dysenteriae type-1 vaccine SC599, attenuated by deletion of invasion (icsA), iron chelation (ent, fep) and shiga toxin A-subunit (stxA) genes, followed by ciprofloxacin on day 4. Dose-independent diarrhea or change in bowel habit was seen in 3 subjects, without dysentery, vaccinaemia or serious adverse events. Hematology and biochemical parameters were unchanged. Doses of 10(5) or greater induced dose-independent SD1 lipopolysaccharide-specific antibody secreting cell (ASC) responses. Geometric mean number of IgA ASCs per 10(6) PBMCs for 10(5), 10(6), 10(7) and 10(8) groups were respectively 41, 8.8, 26 and 8.5. Serum antibody responses were seen in three subjects. SC599 appears immunogenic with maximum tolerated dose greater than 10(8)CFU.

Comparative Immunogenicity of HIV-1 gp140 Vaccine Delivered by Parenteral, and Mucosal Routes in Female Volunteers; MUCOVAC2, A Randomized Two Centre Study

Background Defining optimal routes for induction of mucosal immunity represents an important research priority for the HIV-1 vaccine field. In particular, it remains unclear whether mucosal routes of immunization can improve mucosal immune responses. Methods In this randomized two center phase I clinical trial we evaluated the systemic and mucosal immune response to a candidate HIV-1 Clade C CN54gp140 envelope glycoprotein vaccine administered by intramuscular (IM), intranasal (IN) and intravaginal (IVAG) routes of administration in HIV negative female volunteers. IM immunizations were co-administered with Glucopyranosyl Lipid Adjuvant (GLA), IN immunizations with 0.5% chitosan and IVAG immunizations were administered in an aqueous gel. Results Three IM immunizations of CN54 gp140 at either 20 or 100 μg elicited significantly greater systemic and mucosal antibodies than either IN or IVAG immunizations. Following additional intramuscular boosting we observed an anamnestic antibody response in nasally primed subjects. Modest neutralizing responses were detected against closely matched tier 1 clade C virus in the IM groups. Interestingly, the strongest CD4 T-cell responses were detected after IN and not IM immunization. Conclusions These data show that parenteral immunization elicits systemic and mucosal antibodies in women. Interestingly IN immunization was an effective prime for IM boost, while IVAG administration had no detectable impact on systemic or mucosal responses despite IM priming. Clinical Trials Registration EudraCT 2010-019103-27 and the UK Clinical Research Network (UKCRN) Number 11679

A Single Dose of Oral BCG Moreau Fails to Boost Systemic IFN-γ Responses to Tuberculin in Children in the Rural Tropics: Evidence for a Barrier to Mucosal Immunization.

Immune responses to oral vaccines are impaired in populations living in conditions of poverty in developing countries, and there is evidence that concurrent geohelminth infections may contribute to this effect. We vaccinated 48 children living in rural communities in Ecuador with a single oral dose of 100 mg of BCG Moreau RDJ and measured the frequencies of tuberculin-stimulated peripheral blood mononuclear cells expressing IFN-γ before and after vaccination. Vaccinated children had active ascariasis (n = 20) or had been infected but received short- (n = 13) or long-term (n = 15) repeated treatments with albendazole prior to vaccination to treat ascariasis. All children had a BCG scar from neonatal vaccination. There was no evidence of a boosting of postvaccination IFN-γ responses in any of the 3 study groups. Our data provide support for the presence of a barrier to oral vaccination among children from the rural tropics that appeared to be independent of concurrent ascariasis.