A phase I/II study to evaluate safety, tolerability and immunogenicity of Hillchol®, an inactivated single Hikojima strain based oral cholera vaccine, in a sequentially age descending population in Bangladesh.

BACKGROUND: The World Health Organization (WHO) recommends the use of oral cholera vaccines (OCVs) as part of an integrated control program, both in highly endemic settings and during cholera epidemics. The available and internationally recommended WHO-prequalified OCVs (Dukoral, Shanchol, Euvichol) contain multiple heat and formalin-killed V. cholerae strains of Inaba and Ogawa serotypes. MSD Wellcome Trust Hilleman Laboratories Pvt. Ltd. in technical collaboration with University of Gothenburg, Sweden has developed a new single strain OCV, Hillchol. This vaccine consists of formaldehyde-inactivated whole cell El Tor V. cholerae O1 bacteria engineered into the Hikojima serotype for stable expression of both the Ogawa (AB) and Inaba (AC) LPS antigens on the bacterial surface. We evaluated the safety and immunogenicity of this novel and potentially much less expensive OCV in comparison with Shanchol. METHODS: We conducted a randomized, non-inferiority, age-descending clinical trial of OCV (Hillchol vs. Shanchol) in the Mirpur area of Dhaka city from July 2016 to May 2017. This study was carried out in three different age cohorts (1-<5, 5-17 and ≥18 years old). Two doses of vaccine were given at 14 days intervals to 560 healthy participants. FINDINGS: No serious adverse events were reported. There were no significant differences in the rates of adverse events between the test vaccine (Hillchol) and the comparator (Shanchol) group. Serum vibriocidal antibody responses in all age groups combined were comparable for all the O1 Ogawa (59% vs. 67%; 90% CI of difference: -14.55, -0.84) and Inaba (70% vs. 71%; 90% CI of difference: -7.24, 5.77) serotypes, showing that the Hillchol vaccine was non-inferior to Shanchol. This new vaccine was also non-inferior to Shanchol in the different age strata. CONCLUSION: The safety and immunogenicity profile of the new OCV Hillchol is comparable to Shanchol in persons residing in a cholera-endemic setting. ClinicalTrials.gov number: NCT02823899.

Development of Hillchol®, a low-cost inactivated single strain Hikojima oral cholera vaccine.

Cholera remains an important global health problem with up to 4 million cases and 140,000 deaths annually. Oral cholera vaccines (OCVs) are now a cornerstone of the WHOs "Ending Cholera - A Global Roadmap to 2030" global program for the eventual elimination of cholera. There are currently three WHO prequalified OCVs available, Dukoral®, Shanchol® and Euvichol-Plus®. These vaccines are effective but due to a multiple strain composition and two different methods of inactivation, are complex and costly to manufacture. We describe here the characterization and industrial scale development of Hillchol®; a novel, likely affordable single-component OCV for low and middle-income countries. Hillchol® consists of formalin-inactivated bacteria of a stable recombinant Vibrio cholerae O1 El Tor Hikojima serotype strain expressing approximately 50% each of Ogawa and Inaba O1 LPS antigens. The novel OCV can be manufactured on an industrial scale at a low cost. Hillchol® was well tolerated in animal toxicology studies and shown to have non-inferior oral immunogenicity in mice for both intestinal-mucosal and serological immune responses when compared with a WHO-prequalified OCV. The optimized production of this single component OCV will reduce cost of OCV production and thus substantially increase vaccine availability. Based on these results, Hillchol® has been produced at a GMP facility and used successfully for clinical phase I/II studies.

A randomized Phase I/II study to evaluate safety and reactogenicity of a heat-stable rotavirus vaccine in healthy adults followed by evaluation of the safety, reactogenicity, and immunogenicity in infants.

Objectives: To assess the safety and reactogenicity of single oral dose of heat-stable rotavirus vaccine (HSRV) in healthy adults aged 18-45 years followed by assessment of safety, reactogenicity, and immunogenicity of three doses of HSRV in healthy infants aged 6-8 weeks at enrollment.Trial Design: Single-center randomized controlled, sequential, blinded (adults) and open-label (infants).Setting: Single site at International Center for Diarrheal Disease Research, Bangladesh (icddr,b).Participants: Fifty eligible adults randomized in 1:1 ratio (HSRV: Placebo) followed by 50 eligible infants randomized in 1:1 ratio (HSRV: Comparator (RotaTeq®, pentavalent human-bovine (WC3) reassortant live-attenuated, rotavirus vaccine)).Intervention: Adults received either a single dose of HSRV or placebo and followed for 14 days. Infants received three doses of either HSRV or comparator with a follow-up for 28 days after each dose.Main Outcome Measures: Solicited and unsolicited adverse events (AEs) along with any serious adverse events (SAEs) were part of the safety and reactogenicity assessment in adults and infants whereas serum anti-rotavirus IgA response rates were part of immunogenicity assessment in infants only. Post-vaccination fecal shedding of vaccine-virus rotavirus strains was also determined in adults and infants.Results: In this study, HSRV, when compared with placebo, did not result in increase in solicited adverse events (solicited AEs) in adults. In infants, HSRV had a safety profile similar to comparator vis-à-vis solicited AEs. In infants, fecal shedding of vaccine-virus strains was not detected in HSRV recipients but was observed in two comparator recipients. Percentage of infants exhibiting threefold rise in serum anti-rotavirus IgA titers from baseline to 1-month post-dose 3 in HSRV group was 88% (22/25) and 84% (21/25) in comparator group.Conclusion: HSRV was found to be generally well-tolerated in both adults and infants and immunogenic in infants.

The immunogenic characteristics associated with multivalent display of Vi polysaccharide antigen using biodegradable polymer particles.

Polysaccharides in their great majority are thymus-independent (TI) antigens. Anti-polysaccharide antibody responses are generally weak and characterized by lack of memory, isotype restriction and delayed ontogeny. We report here the generation of protective memory antibody response by multivalent display of polysaccharide antigens on biodegradable polymeric particles. Single dose immunization using polylactide (PLA) polymer particles entrapping Vi capsular polysaccharide antigen from Salmonella typhi promoted isotype switching and induced polysaccharide-specific memory antibody response in experimental animals. PLA nanoparticles as well as microparticles entrapping Vi polysaccharides elicited high IgG titer in comparison to soluble Vi immunization. Immunizations with particles co-entrapping both Vi polysaccharide and tetanus toxoid did not improve the anti-polysaccharide antibody responses. Lower antibody response from co-entrapped formulation was mostly due to inhibition of particle phagocytosis by the macrophages. Immunization using polylactide particles entrapping only Vi polysaccharide with higher density on surface elicited highest secondary antibody response as well as promoted isotype switching. The vaccination potential of particle based immunizations was further confirmed by the generation of quick memory antibody responses while challenging the immunized animals with live S. typhi. This approach provides a multivalent display of polysaccharide antigen using polymer particles and elicits protective memory antibody response without conjugation to a carrier protein.

Systems biology of vaccination for seasonal influenza in humans.

Here we have used a systems biology approach to study innate and adaptive responses to vaccination against influenza in humans during three consecutive influenza seasons. We studied healthy adults vaccinated with trivalent inactivated influenza vaccine (TIV) or live attenuated influenza vaccine (LAIV). TIV induced higher antibody titers and more plasmablasts than LAIV did. In subjects vaccinated with TIV, early molecular signatures correlated with and could be used to accurately predict later antibody titers in two independent trials. Notably, expression of the kinase CaMKIV at day 3 was inversely correlated with later antibody titers. Vaccination of CaMKIV-deficient mice with TIV induced enhanced antigen-specific antibody titers, which demonstrated an unappreciated role for CaMKIV in the regulation of antibody responses. Thus, systems approaches can be used to predict immunogenicity and provide new mechanistic insights about vaccines.

Controlled release of bioactive recombinant human growth hormone from PLGA microparticles.

Controlled release formulation of recombinant human growth hormone (r-hGH) was achieved using poly lactide-co-glycolide (PLGA) polymer. Denaturation of r-hGH by dichloromethane during primary emulsification step of particle preparation was minimized by using human serum albumin whereas inclusion of sucrose and sodium bicarbonate helped in reducing protein denaturation during lyophilization and polymer particle degradation. Encapsulation efficiency of r-hGH entrapped in PLGA particles (size approximately 30 microm) was around 45% with protein load 20 microg of r-hGH/mg of polymer particles. Porous particles showed quick release of r-hGH in comparison to non-porous particles in vitro. More than 10 ng/mL of bioactive r-hGH was found in the serum of the experimental animals observed for a 30-day period after a single intramuscular injection of the polymeric formulation. Incorporation of optimal stabilizers is thus essential for the development of a stable, month long controlled release of polymer particle based r-hGH formulation.

Memory antibody response from antigen loaded polymer particles and the effect of antigen release kinetics.

Memory antibody response is the hallmark of long lasting immunity. In this study, we report the generation of memory antibody response while immunizing with single dose of polymer particle entrapped antigens. Immunization with admixture of alum and polylactide (PLA) polymer particles (2-8 microm) entrapping antigens not only elicited long lasting primary antibody response but also very high levels of memory antibody titer upon re-exposure to very small amount of soluble antigen. In the case of tetanus toxoid (TT), the memory antibody titers from PLA particle based immunization were almost four times higher than that achieved from two doses of alum adsorbed antigen and sustained at a higher level for a longer period of time. Memory antibody response was detected even after challenging the animals after 18 months of primary immunization. Similar enhanced memory antibody response was also observed in the case of immunization with PLA particle entrapping diphtheria toxoid (DT). Memory antibody response generated from polymeric formulations was highly antigen specific. Polymer particles with different release profile of antigen were used as a model system to evaluate the role of antigen on immunological memory. The results suggest that slow and continuous release of antigen from polymer particles plays a critical role in eliciting improved memory antibody response from single point immunization.

Role of alum in improving the immunogenicity of biodegradable polymer particle entrapped antigens.

This study was aimed at understanding the role of alum in improving the immunogenicity of biodegradable polymer particle entrapped antigens. Presence of alum formed a fine network around PLA particles holding them together and promoted attachment of higher number of particles on macrophage surface for a considerable period of time. Use of alum lowered the burst release of the entrapped antigen from particles and thereafter also reduced the cumulative release of antigen from particles. Apart from this, PLA microparticles alone induced macrophages to release TNF-alpha similar to that induced by alum. However admixture of PLA particles and alum enhanced the secretion of TNF-alpha from 876pg/ml at 6h to 3500pg/ml at 24h which was higher than that induced by alum adsorbed TT. Immunization with admixture of antigen loaded polylactide (PLA) microparticles (2-8microm) and alum improved the antibody titers almost twice than that achieved from particle alone in experimental animals. Single point immunization with particle entrapped antigens and alum also elicited antibody titers comparable to two doses of alum adsorbed tetanus toxoid (TT) or diphtheria toxoid (DT). Our results suggest that presence of alum acts in multiple ways to improve the antibody titers of polymer particles entrapped antigens. Such co-operative adjuvant action of alum and polymer particles can be exploited to improve the immunogenicity of other antigens.

Evaluation of parenteral depot insulin formulation using PLGA and PLA microparticles.

PLGA and PLA microparticles entrapping insulin are prepared by solvent evaporation method and are evaluated in diabetes-induced rat for its efficacy in maintaining blood sugar level from a single intramuscular dose. In vitro release of insulin from PLGA and PLA microparticles are 75.35 +/- 1.73% and 67.536 +/- 2.23%, respectively in 168 h (7 days). Released insulin from polymer particles are mostly in monomeric form without aggregation. Optimal use of stabilizers during particle formulation helps in reducing protein denaturation and thus results in stabilized insulin-loaded polymer particles. Intramuscular administration of insulin-loaded PLGA (50 : 50) and PLA microparticles (equivalent to 25 IU insulin/kg of animal weight) in alloxaninduced diabetic rats result in 53.86 +/- 4.2% and 39.52 +/- 6.7% reduction in blood glucose level, respectively in 96 h. This effect continued up to 7 days in case of PLGA and PLA microparticles.

Interactions of antigen-loaded polylactide particles with macrophages and their correlation with the immune response.

Size of the polymeric particulate antigen delivery system and its interactions with antigen-presenting cells (APCs) influence the immune response both qualitatively and quantitatively. In this paper, we report that antigen-loaded polymeric microparticles elicit antibody titers without being phagocytosed by macrophages; and size of the antigen-loaded particles modulates immune response from single-point immunization. Antibody titers varied significantly from single-point immunization with different sized polylactide (PLA) particles entrapping hepatitis B surface antigen. Nanoparticles (200-600 nm) were efficiently taken up by macrophages and elicited lower antibody titers in comparison to microparticles (2-8 microm). PLA microparticles that elicited highest and long-lasting antibody titers from single-point immunization were not taken up by the macrophages and found attached to the surface of the macrophages. Immunization with nanoparticles (200-600 nm) was associated with higher levels of IFN-gamma production, upregulation of MHC class I molecules along with antibody isotypes favoring Th1-type immune response. Immunization with microparticles (2-8 microm size) promoted IL-4 secretion, upregulated MHC class II molecules and favored Th2-type immune response. Western blot analysis showed that release of HBsAg from surface-attached microparticles into macrophages increased with time, but was more or less constant in case of nanoparticles. Our results suggest that continuous release of high concentration of antigen from cell surface-attached PLA microparticles into APCs results in improved antibody response from single-point immunization. It also offers an exciting possibility of designing size-based polymer particle delivery system to modulate immune response.