Analysis of novel meningococcal vaccine formulations

ABSTRACT

The protective effect of meningococcal vaccines targeting disease causing serogroups exemplified by the introduction of MenAfriVac™ in Africa, is well established and documented in large population-based studies. Due to the emergence of other meningococcal disease causing serogroups, novel vaccine formulations are needed. There is a high potential for novel nanotechnology-based meningococcal vaccine formulations that can provide wider vaccine coverage. The proposed meningococcal vaccine formulation contains spherical shaped micro and nanoparticles that are biological mimics of Niesseria meningitidis, therefore present to immune system as invader and elicit robust immune responses. Vaccine nanoparticles encapsulate meningococcal CPS polymers in a biodegradable material that slowly release antigens, therefore enhance antigen presentation by exerting antigen depot effect. The antigenicity of meningococcal vaccine delivered in nanoparticles is significantly higher when compared to vaccine delivered in solution. Preclinical studies are required to assess the immunogenicity of novel vaccine formulations. Therefore, implementing various in-vitro human immune cell-based assays that mimic in-vivo interactions, would provide good insight on optimal antigen dose, effective antigen presentation, facilitate screening of various vaccine and adjuvant combinations and predict in-vivo immunogenicity. This rapid approach is cost-effective and provides data required for the preclinical immunogenicity assessment of novel meningococcal vaccine formulations.

KEYWORDS:

• antigen presentation
• autophagy
• capsular polysaccharide
• CD95
• FAS
• innate immune responses
• macrophage
• meningococcal disease
• neisseria meningitidis
• vaccine

The potential for novel meningococcal vaccine formulations

Neisseria meningitidis is the leading cause of meningitis and meningococcaemia world-wide.¹ Meningococcal capsular polysaccharides (CPS) are major virulence factor and form the basis of preventive vaccines.² To date, there are twelve distinct meningococcal serogroups designated based on the composition of CPS polymers, with serogroups A, B, C, W, Y and X are responsible for the majority of disease.³ The protective effect of meningococcal vaccines targeting disease causing serogroups is well established as demonstrated in large population-based studies.⁴ The introduction of meningococcal serogroup A conjugate vaccine (MenAfriVac®) to Africa's meningitis belt countries led to a significant reduction of disease burden,^5,6 and induced herd immunity effect.⁷ The current polysaccharide meningococcal vaccines that cover other disease causing serogroups are available and very expensive. These vaccines are Menactra™ (Sanofi Pasteur quadrivalent meningococcal conjugate MCV4 vaccine), Nimenrix® (GlaxoSmithKline quadrivalent conjugate meningococcal vaccine) and Menveo® (Novartis quadrivalent conjugate meningococcal vaccine).^8,9 Menactra™ (MCV4 is a quadrivalent conjugate vaccine) and Menomune® (MPSV4 is a quadrivalent polysaccharide vaccine) both contain serogroup A, C, W135 and Y. The commercially available meningococcal vaccines are conjugated to a protein carrier and do not contain adjuvants in their formulation.⁹ These licensed meningococcal conjugate vaccines require booster doses to maintain a protective immune response,^10,11 and needle use for administration. The high cost of chemical conjugation, the cold-chain (continuous refrigeration) requirement, and single dose preparation are limitation of producing and distributing conjugate vaccines. These limitations are inherent to all currently licensed vaccines.

The development of effective vaccine and adjuvant formulations depends on the sustained release of immunogens and their consequent uptake by professional immune cells. Successful vaccines elicit innate immune responses that lead to optimal adaptive immune responses.^2,12 A key factor in inducing robust immune responses is the adequate presentation by antigen presenting cells (APCs) like dendritic cells over a sustained time interval. The recent advances in the field of nanotechnology facilitated the development of novel meningococcal vaccine formulation that mimics conjugation effects by encapsulation into albumin-based nanoparticle matrices.^8,13,14 The spherical shaped micro and nanoparticles are biological mimics of N. meningitidis bacteria, therefore present to immune system as invader but without the ability to cause disease. The novel meningococcal vaccine nanoparticles formulation encapsulates meningococcal CPS polymers in a biodegradable material that slowly release antigens, therefore enhance antigen presentation by exerting antigen depot effect. The antigenicity of meningococcal vaccine delivered in nanoparticles is significantly higher when compared to vaccine delivered in solution.¹³ In addition, the antigenicity of meningococcal vaccine nanoparticles formulation is greatly enhanced when combined with adjuvants like Alum or MF59 also encapsulated in nanoparticles.⁸ These adjuvants are approved by Federal and Drug Administration (FDA) to be used with licensed vaccines. There is a high potential for novel nanotechnology-based vaccines formulations that slowly release antigens and act as antigen depot to enhance antigen presentation, boost immune responses and overcome the limitations of traditionally formulated vaccine antigens. The novel meningococcal vaccine nanoparticles formulation is inexpensive and can be stored as a dry powder with extended shelf life that does not require the cold-chain which facilitates storage and distribution.

This novel nanotechnology-based vaccine formulation would be suitable for meningococcal vaccine delivery in sub-Saharan Africa. The introduction of MenAfriVac™ (meningitis A vaccine), led to a dramatic reduction in meningococcal disease caused by this serogroup.¹⁵ As expected, other meningococcal disease-causing serogroups emerged in Africa like serogroup W and X which is a considerable concern and advocates for a wider vaccine serogroups coverage.^16,17 In addition to persistent low carriage of meningococcal serogroup A, other serogroups like Y and X are causing meningococcal infections in sub-Saharan Africa.^7,18-20 Therefore, designing novel meningococcal vaccine nanoparticles formulations that cover emerging and persistent meningococcal disease-causing serogroups with enhanced antigenicity have advantages over currently used conjugate vaccines. The novel nanotechnology-based formulations will provide suitable solution not just to low resource countries with endemic and heavy disease burden but also suitable for vaccinations required for the annual Hajj season.¹

The major hurdle in vaccine licensing is providing vaccine efficacy and biosafety data which requires clinical trials and incurs great expense, time and effort. Preclinical studies that precede costly clinical trials are essential to assess the immunogenicity of novel vaccine formulations. Therefore, implementing various in-vitro cell-based assays that mimic in-vivo interactions, would provide good insight on optimal antigen dose, effective antigen presentation, and facilitate screening of various vaccine and adjuvant combinations. This rapid approach is cost-effective and provides data required for the preclinical immunogenicity assessment of novel meningococcal vaccine formulations.

Antigenicity profiling of meningococcal vaccine formulations using in vitro cell-based assays

Since Neisseria meningitidis is a strictly human pathogen, using animal models to investigate the immunogenicity of meningococcal vaccines has a major caveat. Alternatively, the immunogenicity of novel vaccine formulations can be assessed using human immune cells in multiple in-vitro cell-based assays. The collective results of vaccine-induced immune responses generated using in-vitro cell-based assays are good indicators of vaccine bioactivity and predict vaccine immunogenicity in-vivo. These in-vitro human immune cell-based assays have several advantages when compared to in-vivo animal models such as rapid results, reduced cost, effort, animal use ethics and species-specific differences that may skew immunogenicity data. Technically, human monocyte derived cells can serve as antigen presenting cells (APCs) when pulsed with various doses of vaccine antigens in a tissue culture plate scaled down to a 96-well microtiter plate as previously described.^8,13,14 An array of innate immune responses can be measured including cytokines release, phagocytosis, autophagy induction, cellular viability and apoptosis, as well as the expression of dendritic cell maturation and antigen presentation markers MHC I, MHC II, CD40, CD80, CD83 and CD86.^8,13,14,21

Meningococcal vaccine-induced innate immune responses

Neisseria meningitidis capsular polysaccharides (CPS) that form the basis of vaccine, are recognized via TLR2 and TLR4-MD-2 and elicit the release of cytokines and chemokines from macrophages.^2,22,23 The ability of vaccine antigens to induce innate immune responses as a prerequisite of inducing adaptive immunity can be examined using in-vitro cell-based assays.^8,13,14,21 The experimental meningococcal vaccine nanoparticles have a spherical shape with size ranges between 500 to 1000 nm, therefore mimic invading pathogens without the ability to cause disease. Upon contact with antigen presenting cells like macrophages or dendritic cells, vaccine nanoparticles induce phagocytosis which then triggers the respiratory burst leading to reactive oxygen species (ROS) release in large amounts. ROS not only potentiate the oxidative killing of invading pathogens but also play an important role as a second messenger that consequently enhances adaptive immune responses.²⁴ We previously reported that the uptake of vaccine nanoparticles by macrophages induced cytokines and chemokines release and enhanced phagocytosis-induced ROS release.^8,13,14 The most common reactive species released are superoxide anions and hydrogen peroxides that lead to the formation of hypochlorous acid which then acts as a natural adjuvant by facilitating antigen processing, cross-priming and the induction of adaptive immune responses.²⁵ Therefore, meningococcal vaccine nanoparticles induced phagocytosis-dependent ROS release enhance antigen uptake and subsequently influence antigen processing and loading on MHC-II.²⁶

Upon uptake of vaccine nanoparticles, immune cells induce autophagy process to degrade and process the foreign cargo.²⁷ The important role of autophagy induction in antigen presentation and adaptive immune responses has been established.^28,29 We previously reported that meningococcal vaccine nanoparticles induced dose-dependent autophagy in macrophages.¹⁴ The observed large cytosolic autophagic vacuoles are induced by vaccine nanoparticles but not by empty nanoparticles. Further, meningococcal vaccine in solution, not encapsulated in nanoparticles, induced significantly less autophagic vacuoles in macrophages. Thus, the robust induction of autophagy by vaccine nanoparticles indicates the slow release of antigen from the nanoparticles matrix which is highly desired for optimal vaccine induced responses. Technically, autophagy induction can be assessed using APCs stably transfected with the GFP labeled autophagy marker LC3,^14,27 or by using an autophagy flux staining probe as previously described.³⁰ The slow release of antigen is important for priming immune cells and inducing potent adaptive immune responses.³¹ The kinetics of slow antigen release prolong the time of antigen exposure, recognition and increase immunostimulatory activity, consequently enhancing vaccine antigenicity. The currently available meningococcal vaccines contain CPS antigens in solution, therefore, meningococcal vaccine nanoparticles formulations are expected to induce better antigen presentation, hence stronger adaptive immune responses.

Effective antigen presentation assessment in-vitro

Efficacious vaccines induce a combination of protective humoral and cellular adaptive immune responses. For meningococcal disease, the complement-mediated killing of N. meningitidis and serum opsonic activity confer protection.^9,32 Upon vaccination APCs activate naïve T lymphocytes by direct contact to induce adaptive immune responses.³³ Activated APCs demonstrate increased surface expression of antigen presenting molecules MHCI and MHCII as well as co-stimulatory molecules CD80 and CD86 that bind to their receptors TCR and CD28 respectively, expressed on the surface of naïve T cells.³³ This direct interaction activates naïve T cells and induce proliferation which is a prerequisite to elicit protective adaptive immune response including B cell activation and differentiation leading to humoral antibody response.^9,33 This process of T cell activation can be mimicked in-vitro using cell-to-cell contact (APCs-T cells) assays, where APCs pulsed with vaccine antigen are co-incubated with naïve T cells.^8,21 The activation and proliferation of T lymphocytes can be monitored using FACS analysis tracing the fluorescence peaks of CellTracer™ (fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester CFSE) dye.³⁴ Human immortalized cell lines that are commercially available from the American Tissue Culture Collection (ATCC) such as THP-1 monocytic cells and Jurkat T lymphocytic cells, as well as Toledo B lymphocytes, can be employed in APCs-T cell contact model to provide preclinical data on vaccine antigenicity. In addition, primary human peripheral monocytic cells and T lymphocytic cells obtained from healthy donors can be used to assess vaccine immunogenicity profile in-vitro which confirm or recapitulate data generated using immortalized cell lines.³⁴ These assays are not difficult to perform with well-established experimental protocol; can be tailored to address specific vaccine formulation issues; can be scaled down into 96-well format and are reproducible. These in-vitro cell-based assays provide data on effective antigen presentation as assessed by the expression of antigen presenting MHCI and MHCII, and co-stimulatory molecules CD80, CD86, CD40, as well as T cell activation and proliferation. In-vitro cell-based assays are also commercially available and currently employed by pharmaceutical companies.

Monitoring CD95 (FAS) expression in vitro

Effective antigen presentation depends on the viability of antigen presenting cells. Some vaccine antigens have the ability to induce the death receptor CD95 (FAS) expression on the surface of APCs which triggers apoptosis leading to reduced antigen presentation, consequently suboptimal adaptive immune responses.^13,35-39 Suboptimal immune responses mediated by CD95 (FAS) activation is reported in human protozoan parasite Trypanosoma cruzi.⁴⁰ We reported that meningococcal CPS serogroup A vaccine induced CD95 (FAS) expression on the surface of APCs in a dose-dependent manner, assessed by Flow cytometry and gene expression.¹³ The high dose of meningococcal CPS-loaded nanoparticles, but not empty nanoparticles, induced the expression of death receptor CD95 (FAS) leading to reduction in cell viability and reduced innate immune responses including TNF-α release.¹³ Therefore, monitoring the expression of CD95 on the surface of APCs using in-vitro cell-based assay would allow screening of various vaccine antigens and adjuvant combination and would predict the ability of vaccine antigens to trigger death pathways. Monitoring CD95 expression would facilitate vaccine dose titration and optimization and help to design vaccines that capable of eliciting optimal protective immunity. Capsular polysaccharides found in encapsulated bacterial pathogens are weak antigens and incapable of demonstrating a T cell dependent immune response, therefore CPS-based vaccines are conjugated with proteins or toxoids to stimulate a stronger immune response to vaccines.⁴¹ B lymphocytes can serve as APCs and respond to vaccine antigens in a T cell-independent manner.^33,41 Meningococcal CPS-based vaccines are known to activate polyclonal B lymphocytes,⁴² however, it is not known whether these activated B lymphocytes express CD95 on their surface. Further, CD95 expression in memory B lymphocyte subset has been reported to correlate with systemic lupus erythematosus disease activity and proposed as a biomarker for this autoimmune disease.^43,44 Therefore, CD95 high expression seems to associate with dysfunctional or inappropriate immune responses. It is tempting to postulate that inability of CPS-based vaccines to induce T cell dependent response in part, may be attributed to their ability to induce the death receptor CD95 (FAS) leading to in-effective antigen presentation. The ability of novel vaccine formulations to induce CD95 expression should be monitored and harnessed to fine tune desired adaptive immune responses to vaccines.

Disclosure of potential conflicts of interest

Author declares no conflict of interest.