Immunocartography: Charting vaccine-driven immunity by applying single cell proteomics to an in vitro human model.

The ability to measure immunomodulatory effects of a vaccine is crucial for novel vaccine design. While traditional animal models have been effective, a better understanding of the response in humans to new vaccines in pre-clinical development is critical for advancement to clinical trials. A translational methodology that can capture the complexity of a vaccine-driven response in a human model, which does not require human exposure, is needed. Here we have designed a platform that uses fresh human whole blood as a key component to study the adaptive immune memory response to vaccine formulations. The response is monitored by high-parameter single cell analysis using mass cytometry (Helios, CyTOF System), allowing for a rapid, in-depth characterization of antigen specific proliferation and expansion of preexisting memory T cells in concert with an innate adjuvant-driven response. In this work we demonstrate the capability of this platform to characterize biologically relevant changes in the cellular response across memory T-cells, B cells, monocytes, and NK cells, at an unprecedented level of detail. This approach that we call Immunocartography has the potential to transform the way new vaccines can be assessed before and throughout clinical development.

A single immunogenicity assay for testing potency of combination DTaP vaccines: Simultaneous quantitation of anti-DT, anti-TT, anti-PTxd and anti-FHA antibodies in Guinea-pig serum with a Luminex®-xMAP® bead-based serological assay.

The diphtheria toxoid (DT), tetanus toxoid (TT), and acellular pertussis (aP) single immunogenicity assay (DTaP SIA) is a Luminex®-xMAP®-bead-based multiplex immunoassay for estimating the potency of DTaP pediatric combination vaccines in guinea pigs. This manuscript describes the validation of this assay for the simultaneous quantitation of anti-diphtheria toxoid (anti-DT), anti-tetanus toxoid (anti-TT), anti-pertussis toxoid (anti-PTxd), and anti-filamentous hemagglutinin (anti-FHA) antibodies in guinea pig serum following injection of a DTaP vaccine formulation. The results were expressed in arbitrary units/mL (AU/mL) using reference serum for comparison. Specificity was demonstrated by ≥ 75% homologous and ≤25% heterologous inhibition for all the antigens. The results were linear for anti-DT, anti-TT, anti-PTxd and anti-FHA antibodies. Accuracy was demonstrated with recovery of between 80% and 120% for all four antibodies. The relative standard deviation of repeatability was ≤20%. The results demonstrate that this SIA can be used for the linear, accurate, and precise simultaneous detection of all four antibodies, based on both the ICH Q2 and the EMA guidelines on bioanalytical method validation.

Preparation and characterization of microencapsulated DwPT trivalent vaccine using water soluble chitosan and its in-vitro and in-vivo immunological properties.

The paper explained the microencapsulation of three different antigenic materials viz. Diphtheria toxoid (DT), whole cell pertussis antigens (PT and FHA) and tetanus toxoid (TT) by coacervation method using water soluble chitosan as a polymer crosslinked by vanillin/TPP co-crosslinkers for the development of oral trivalent DwPT vaccine. Instrumental characterization of chitosan microspheres suggested specific interaction with vanillin/TPP, higher thermal stability, amorphous nature, spherical morphology with size less than 2µm along with positive charge density offering mucoadhesive properties. Furthermore, PT and FHA showed higher encapsulation up to 94% followed by TT and DT. Cumulative release rate of DT was (68.47%), TT (73.67%), PT (43%) and FHA (53%). Release kinetics interpreted using DD solver program, indicated protein release followed first order kinetics and obeyed Korsmeyer-peppas model, stating fickian diffusion relates to diffusion, erosion and controlled release rate of the encapsulated toxoids. Application of formulations on caco-2 cell line showed negligible cytotoxic effect and efficient uptake of FITC labelled microspheres. The obtained in-vivo results suggests that the final trivalent DwPT formulation were having successful elicitation of both systemic (IgG) and mucosal (sIgA) immune response in balb/c mice. Overall studies indicated that DwPT formulation could be a suitable alternative to available injectable DaPT vaccine.

[PROFILES OF CYTOKINES IN MICE DURING IMMUNIZATION WITH ADTP- VACCINE WITH ACELLULAR PERTUSSIS COMPONENT].

AIM: Study cytokine status in mice immunized with vaccines containing acellular pertussis component. MATERIALS AND METHODS: Vaccines developed in Mechnikov RIVS - acellular pertussis vaccine (aPV) and adsorbed pertussis-diphtheria-tetanus vaccine (aDTaP), containing a complex of protective antigens of pertussis microbe - were used in the study. F1 (CBAxC57B16) line mice weighing 12 - 14 g were immunized intraperitoneally 3 times at an interval of 7 days with aPV and aDTaP at human immunization dose (0.5 ml), containing 25 µg of pertussis component. Intact mice were used as a control group. Levels of IFN-,γ, IL-2, IL-4, IL-5, IL-12 cytokines were de- termined after each immunization in enzyme immunoassay using commercial test-systems from Cusabio (China). RESULTS: An increase of levels of IFN-γ, IL-2, IL-5, IL-12 and lack of stimulation of production of IL-4 was established in dynamics of immune response after administration of aPV and aDTaP vaccines. CONCLUSION: The data obtained indicate that immunization of mice with aPV and aDTaP vaccines resulted in activation of production of cytokines characteristic for im- mune response during pertussis infection and immunization with whole-cellular aDTP-vaccines.

Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model.

Pertussis is a highly contagious respiratory illness caused by the bacterial pathogen Bordetella pertussis. Pertussis rates in the United States have been rising and reached a 50-y high of 42,000 cases in 2012. Although pertussis resurgence is not completely understood, we hypothesize that current acellular pertussis (aP) vaccines fail to prevent colonization and transmission. To test our hypothesis, infant baboons were vaccinated at 2, 4, and 6 mo of age with aP or whole-cell pertussis (wP) vaccines and challenged with B. pertussis at 7 mo. Infection was followed by quantifying colonization in nasopharyngeal washes and monitoring leukocytosis and symptoms. Baboons vaccinated with aP were protected from severe pertussis-associated symptoms but not from colonization, did not clear the infection faster than naïve animals, and readily transmitted B. pertussis to unvaccinated contacts. Vaccination with wP induced a more rapid clearance compared with naïve and aP-vaccinated animals. By comparison, previously infected animals were not colonized upon secondary infection. Although all vaccinated and previously infected animals had robust serum antibody responses, we found key differences in T-cell immunity. Previously infected animals and wP-vaccinated animals possess strong B. pertussis-specific T helper 17 (Th17) memory and Th1 memory, whereas aP vaccination induced a Th1/Th2 response instead. The observation that aP, which induces an immune response mismatched to that induced by natural infection, fails to prevent colonization or transmission provides a plausible explanation for the resurgence of pertussis and suggests that optimal control of pertussis will require the development of improved vaccines.

[Preclinical studies of an adsorbed diphtheria-tetanus-pertussis vaccine (ADTP-vaccine) with acellular pertussis component].

AIM: Evaluate standardness of antigenic composition of pertussis component, completeness of sorption of pertussis, diphtheria and tetanus components, specific activity and safety of experimental series ofADTP-vaccine with acellular pertussis component (ADTaP-vaccine). MATERIALS AND METHODS: The content of separate antigens (pertussis toxin, filamentous hemagglutinin and agglutinogens 1, 2, 3) in samples of acellular pertussis component of ADTaP-vaccine and completeness of sorption of pertussis component of ADTaP-vaccine were evaluated by using enzyme immunoassay. Completeness of sorption of diphtheria and tetanus components were determined in flocculation reaction and antitoxin-binding reactions, respectively. Protective activity ofADTaP-vaccine was studied in model ofmeningoencephalitis development in mice infected with Bordetella pertussis (strain 18323) neurotropic virulent culture, protective activity oftetanus component - by survival of mice after administration of tetanus toxin, protective activity of diphtheria component - by survival of guinea pigs after administration of diphtheria toxin. Safety of preparations was evaluated in tests of acute and chronic toxicity with carrying out pathomorphologic studies including immature animals. RESULTS: All the studied experimental series ofADTaP-vaccine were standard by content of separate antigens of pertussis microbe. All the ADTaP-vaccine components were completely sorbed on aluminium hydroxide gel. By protective activity ADTaP preparations satisfied the WHO requirements. The preparations were non-toxic in acute and chronic toxicity and did not induce pathomorphologic changes including immature animals. CONCLUSION: Experimental samples of ADTaP-vaccine by specific activity and safety satisfied WHO requirements.

Early Impact of the US Tdap vaccination program on pertussis trends.

OBJECTIVE: To evaluate the impact of the adolescent Tdap (tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine) vaccination program on pertussis trends in the United States. DESIGN: Retrospective analysis of nationally reported pertussis cases, January 1, 1990, through December 31, 2009. SETTING: United States. PARTICIPANTS: Confirmed and probable pertussis cases. Intervention The US Tdap vaccination program. MAIN OUTCOME MEASURE: Rate ratios of reported pertussis incidence (defined as incidence among 11- to 18-year-olds divided by the combined incidence in all other age groups) modeled through segmented regression analysis and age-specific trends in reported pertussis incidence over time. RESULTS: A total of 200 401 pertussis cases were reported in the United States from 1990 to 2009. Overall incidence ranged from 1.0 to 8.8 per 100,000 persons (1991 and 2004, respectively). Slope coefficients (estimated annual rate of change in rate ratios) from segmented regression showed a steady increase in pertussis incidence among adolescents 11 to 18 years old compared with all other age groups before Tdap introduction (slope = 0.22; P < .001), and a steep decreasing trend post introduction (slope = -0.48; P < .001), suggesting a direct impact of vaccination among adolescents. Indirect effects of adolescent vaccination were not observed among infants younger than 1 year. CONCLUSIONS: Changes in pertussis incidence in the United States from 2005 to 2009 revealed a divergence between 11- to 18-year-olds and other age groups, suggesting that targeted use of Tdap among adolescents reduced disease preferentially in this age group. Increased Tdap coverage in adolescents and adults is needed to realize the full direct and indirect benefits of vaccination.

Spotlight on DTPa-HBV-IPV/Hib Vaccine (Infanrix hexa).

Infanrix hexa, administered intramuscularly, is a diphtheria, tetanus, acellular pertussis, hepatitis B (HBV), inactivated poliomyelitis and Haemophilus influenzae type b (Hib) conjugate vaccine, indicated for primary and booster vaccination of infants. Infanrix hexa should be administered as a two- or three-dose primary vaccination course in infants aged < or =6 months, followed by booster vaccination between 11 and 18 months of age, with an interval of at least 6 months between the last dose of primary vaccination and the booster dose. This spotlight reviews the immunogenicity and protective effectiveness, as well as the reactogenicity and safety of Infanrix hexa. Infanrix hexa as primary and booster vaccination was safe and highly immunogenic for all its component toxoids/antigens in infants aged <2 years, regardless of vaccination schedules. Its immunogenicity and safety profiles were generally similar to those of currently available vaccines, the diphtheria, tetanus and acellular pertussis-based pentavalent vaccines plus monovalent HBV or Hib vaccines. In large clinical studies, Infanrix hexa elicited a strong immune response against vaccine toxoids/antigens, as indicated by high seroprotection/seropositivity/vaccine response rates and geometric mean titers. Moreover, antibodies against vaccine toxoids/antigens persisted for up to a mean of approximately 6 years after booster vaccination, and the vaccine induced long-term immune memory against hepatitis B surface antigen and Hib antigen. A strong immune response against Infanrix hexa toxoids/antigens after primary vaccination was also induced in infants who had received a dose of HBV vaccine at birth and in pre-term infants, although the response in the latter group was somewhat lower than that in full-term infants. In addition, when coadministered with other childhood vaccines, the immunogenicity of Infanrix hexa or that of the concomitantly administered vaccine was generally not altered. Hexavalent vaccines, including Infanrix hexa, were protective against invasive Hib disease; Infanrix hexa is also expected to be protective against pertussis. Most solicited local and general symptoms with Infanrix hexa were mild to moderate in intensity and the vaccine was associated with few unsolicited adverse events. Available clinical data from more than 10 years' experience with the vaccine suggest that Infanrix hexa as primary and booster vaccination is a safe and useful option for providing protection against the common childhood diseases of diphtheria, tetanus, poliomyelitis, pertussis, hepatitis B and invasive Hib disease.

Safety, tolerability, and immunogenicity of gardasil given concomitantly with Menactra and Adacel.

OBJECTIVES: Multinational phase III trials of a human papillomavirus vaccine, Gardasil, have shown the vaccine to be generally well-tolerated, efficacious, and immunogenic. We evaluated the immunogenicity and safety of Gardasil administered concomitantly with Menactra and Adacel. METHODS: In this open-label study, boys (n = 394) and girls (n = 648) aged 10 to 17 were randomly assigned in a 1:1 ratio as follows: group A (concomitant administration) received a 0.5-mL dose of Gardasil at day 1, month 2, and month 6 and a 0.5-mL dose of Menactra and Adacel on day 1; group B (nonconcomitant administration) received Gardasil at day 1, month 2, and month 6 and Menactra and Adacel at month 1. Antibody levels for all vaccine components were measured. Systemic, injection-site, and serious adverse experiences (AEs) were monitored. RESULTS: Immune responses after concomitant administration of the 3 vaccines were noninferior to nonconcomitant administration. Seroconversion for Gardasil was > or = 99% in both groups A and B. For Menactra and Adacel, concomitant administration of the vaccines was demonstrated to be noninferior to nonconcomitant administration. Concomitant administration was generally well-tolerated. No participants withdrew because of an AE. One serious AE of transient muscular weakness of <24 hours' duration after the third Gardasil injection was reported in group B and was deemed possibly vaccine-related by the investigator. CONCLUSIONS: Overall, concomitant administration was generally well-tolerated and did not interfere with the immune response to the respective vaccines. Concomitant administration should minimize the number of visits required to deliver each vaccine individually, leading to increased compliance and more effective disease prevention.

Commonly used prophylactic vaccines as an alternative for synthetically produced TLR ligands to mature monocyte-derived dendritic cells.

Currently dendritic cell (DC)-based vaccines are explored in clinical trials, predominantly in cancer patients. Murine studies showed that only maturation with Toll-like receptor (TLR) ligands generates mature DCs that produce interleukin-12 and promote optimal T-cell help. Unfortunately, the limited availability of clinical-grade TLR ligands significantly hampers the translation of these findings into DC-based vaccines. Therefore, we explored 15 commonly used preventive vaccines as a possible source of TLR ligands. We have identified a cocktail of the vaccines BCG-SSI, Influvac, and Typhim that contains TLR ligands and is capable of optimally maturing DCs. These DCs (vaccine DCs) showed high expression of CD80, CD86, and CD83 and secreted interleukin-12. Although vaccine DCs exhibited an impaired migratory capacity, this could be restored by addition of prostaglandin E(2) (PGE(2); vaccine PGE(2) DCs). Vaccine PGE(2) DCs are potent inducers of T-cell proliferation and induce Th1 polarization. In addition, vaccine PGE(2) DCs are potent inducers of tumor antigen-specific CD8(+) effector T cells. Finally, vaccine PGE(2)-induced DC maturation is compatible with different antigen-loading strategies, including RNA electroporation. These data thus identify a new clinical application for a mixture of commonly used preventive vaccines in the generation of Th1-inducing clinical-grade mature DCs.

Lipopolysaccharide analogs improve efficacy of acellular pertussis vaccine and reduce type I hypersensitivity in mice.

Pertussis is an infectious disease of the respiratory tract that is caused by the gram-negative bacterium Bordetella pertussis. Although acellular pertussis (aP) vaccines are safe, they are not fully effective and thus require improvement. In contrast to whole-cell pertussis (wP) vaccines, aP vaccines do not contain lipopolysaccharide (LPS). Monophosphoryl lipid A (MPL) and Neisseria meningitidis LpxL2 LPS have been shown to display immune-stimulating activity while exerting little endotoxin activity. Therefore, we evaluated whether these LPS analogs could increase the efficacy of the aP vaccine. Mice were vaccinated with diphtheria-tetanus-aP vaccine with aluminum, MPL, or LpxL2 LPS adjuvant before intranasal challenge with B. pertussis. Compared to vaccination with the aluminum adjuvant, vaccination with either LPS analog resulted in lower colonization and a higher pertussis toxin-specific serum immunoglobulin G level, indicating increased efficacy. Vaccination with either LPS analog resulted in reduced lung eosinophilia, reduced eosinophil numbers in the bronchoalveolar lavage fluid, and the ex vivo production of interleukin-4 (IL-4) by bronchial lymph node cells and IL-5 by spleen cells, suggesting reduced type I hypersensitivity. Vaccination with either LPS analog increased serum IL-6 levels, although these levels remained well below the level induced by wP, suggesting that supplementation with LPS analogs may induce some reactogenicity but reactogenicity considerably less than that induced by the wP vaccine. In conclusion, these results indicate that supplementation with LPS analogs forms a promising strategy that can be used to improve aP vaccines.

Seroepidemiology of pertussis in the adult population of Singapore.

INTRODUCTION: Pertussis is a highly communicable, vaccine-preventable respiratory disease and a frequent but often underestimated cause of prolonged cough illness in adults. Protection after childhood vaccination is minimal after 10 years without boosting. The need for adult booster depends on the national epidemiology. MATERIALS AND METHODS: We did a seroepidemiological survey amongst the adult population (aged 18 to 45 years) of Singapore. None had received pertussis booster vaccine in the preceding 10 years. We measured IgG antibodies to pertussis whole cell antigen. RESULTS: Two hundred and seventy subjects with the median age of 30 years were enrolled. We found positive IgG antibody levels in 97% of the population. Seropositivity was not associated with age, gender or race. CONCLUSION: The seroprevalence in adults was much higher than the previously documented seroprevalence of around 50% in the adolescent age group in Singapore. The increase is most likely due to natural infection with B. pertussis. Pertussis booster vaccine for adolescents/young adults in Singapore would be indicated.

IL-1beta-dependent neurological effects of the whole cell pertussis vaccine: a role for IL-1-associated signalling components in vaccine reactogenicity.

Immunization with the whole cell pertussis vaccine (Pw), but not the acellular pertussis vaccine (Pa), is associated with a number of neurological side effects. Previously, we have demonstrated a role for interleukin-1beta (IL-1beta) in Pw reactogenicity. Here we report that parenteral Pw administration resulted in a concomitant increase IL-1 type I receptor (IL-1RI) mRNA and a decrease in IL-1 type II receptor (IL-1RII) mRNA expression in the murine hypothalamus. These Pw-induced changes were accompanied by an increase in caspase-1 and interleukin-1beta (IL-1beta), and were associated with increased activity of the stress-activated kinase, p38. In contrast, immunization with Pa failed to activate pro-inflammatory IL-1 responses but resulted in increased IL-1 receptor antagonist (IL-1ra) production. These results suggest that the neurological effects of Pw are associated with central activation of IL-1beta and IL-1-associated signalling components.

Interfering effect of diphtheria-tetanus-acellular pertussis combined (DTaP) vaccines on the bacterial endotoxin test.

We evaluated applicability of the endotoxin test to DTaP vaccines. Although no DTaP vaccine batches showed immediate interference with the activating activity of spiked endotoxin on the clotting of Limulus amaebocyte lysate (LAL), a gradually progressing interference depending on the time after spiking was seen for some of the batches. The interfering DTaP vaccine batches, however, showed no significant effect onin vitro TNF-alpha induction in RAW264.7 cells and pyrogenicity in rabbits of spiked endotoxin. Aluminium hydroxide gel in the vaccines was suggested to be one of the causes of the interference. Accordingly, a careful evaluation of the interfering effect was assumed crucial for the effective application of the endotoxin test to check residual biological activity of endotoxin in DTaP vaccines.