Neisseria meningitides or meningococcus - Essay Example

Paper Critique Question Neisseria meningitides, commonly known as meningococcus, is a bacterium that can cause large epidemics of meningitis which is an inflammation of the membranes covering the brain and spinal cord (meninges). Acute bacterial meningitis is a medical emergency that can lead to severe brain damage and cause death especially in children. Meningococcal infection can also cause severe sepsis that could be fatal. Serogroup B meningococcus is one of the twelve groups of meningococci identified so far based on the capsular polysaccharides. No universal vaccine has been developed against this bacterial group to prevent the meningococcal diseases. Generally, the proteins on the surface of a bacterium known as antigens instigate the immune system to produce immunoglobulin proteins called antibodies. The antibodies have bactericidal activity that is, they can kill the bacteria. Such antigens generally constitute the vaccine against the particular bacterium. This paper describes how five antigens of serogroup B meningococcus were discovered by reverse vaccinology, which is a genomics-based technique that identifies, with the help of computer programs, slices of the bacterial genome that potentially code for the microbe’s surface proteins having antigenic activity. The predicted antigenic activities of the proteins are verified in mice after cloning and expression in E.coli, and using the fusion products obtained to immunize mice after purification. The five antigens identified were produced in a form that was suitable for large-scale production and made into vaccine form for human use by adding chemical compounds (adjuvants) that improve the immune response to vaccine antigens. Results showed that when the inorganic compound, aluminium hydroxide was used as the adjuvant, the vaccine elicited antibodies that could kill 78% of the 85 meningococcal strains tested. Strain coverage improved to 90% by using either MF59, a detergent-stabilized oil-in-water emulsion or CpG oligonucleotides activate cells of the immune system as adjuvants. The vaccine, therefore, has significant potential to conquer one of the most devastating diseases in children. Question 2 The vaccine described in this study acts against one serogroup of N. meningitides identified as serogroup B meningococcus (MenB). According to Giuliani et al. (2006), the annual incidence worldwide of bacterial meningitis in children and young adults is nearly 1.2 million. About 32% of meningococcal disease reported in the United States, and between 45% and >80% of meningococcal disease reported in Europe are due to MenB. Tetravalent vaccines made from the purified capsular polysaccharides of serogroups A, C, Y, and W135 have been successfully used to immunize adults for thirty years while conjugate vaccines, for use in all age groups, were developed a decade ago. A universal vaccine for meningococcal meningitis caused by all forms of N. meningitides would eliminate the disease forever. But the development of such a vaccine has been hampered by the presence of extensive variation in the proteins on the surface of the bacterium. The outer membrane proteins, in particular PorA in N. meningitides, is generally the primary target for a vaccine-induced antibody. In nature, the bacterium mutates constantly thereby altering the interactions with antibody which reduces or nullifies the efficacy of the vaccine. Therefore, the development of a universal vaccine has been a huge challenge. Moreover, unlike in the other serogroups of N. meningitides, a capsular polysaccharide of MenB is identical to the polysialic acid [α(2-8) N-acetylneuraminic acid] present in many human glycoproteins (Giuliani et al., 2006). As this makes the polysaccharide–protein conjugate vaccines unviable to combat MenB disease, there has been no universal vaccine available for MenB. Hence, reverse vaccinology had to be used for vaccine development against MenB. Question 3 The sequencing of the whole genome of MenB was necessary in order to submit the organism to reverse vaccinology that would enable identification of novel antigens. As a result of this exercise, twenty eight unique antigens were identified that had the ability to induce antibodies with bactericidal activity (BCA) in mice immunized with the same in Freund’s complete adjuvant (FCA). However, detailed studies on some of the potential antigens yielded a few antigens that were good candidates for a universal vaccine whereas some others such as GNA33 had to be discarded on account of serosubtype specificity as it was found to be a mimotope of PorA. From among the protein–protein fusions generated, the authors were able to formulate a multicomponent vaccine consisting of five antigens, hypothesizing that it would increase the breadth of the vaccine coverage and avoid selection of escape mutants. Four of the five antigens were expressed as fusion proteins of which the most promising combinations to facilitate large-scale industrial manufacturing of the vaccine were found to be GNA2132 with GNA1030 and GNA2091 with GNA1870. FACS as well as BCA assays showed that the immune responses induced by the two fusion proteins adjuvanted with aluminum hydroxide were more potent than those induced by the individual antigens. In comparison to the protein-based vaccines composed of outer membrane vesicles (OMV) with a coverage of 20% of the MenB strains, the 5 component vaccine against MenB or 5CVMB vaccine could induce broader protection without any serosubtype-specificity and covered most of the population diversity (78% of the strains tested using aluminium hydroxide adjuvant and >95% using another adjuvant, MF59 along with CpG oligonucleotides. The study by Giuliani et al. (2006) thus revealed that universal protein-based vaccines can be developed against encapsulated bacteria that are usually targeted by conjugate vaccines. Question 4 The complement-mediated antibody-dependent serum bactericidal activity (BAC) assay was used to determine the efficacy of the 5CVMB vaccine. The principle of this assay is that, under suitable conditions, the antibody recognizes surface-exposed antigens and binds to complement (activation via the classical pathway), resulting in bacteriolysis and death of the target organism. BAC determined in bacteria incubated with complement serum and immune sera is expressed in terms of CFU per milliliter. The methodology for BAC determination as described in an earlier paper by the authors (reference number 23, Giuliani et al., 2006) is as follows: N. meningitidis strains are grown overnight on chocolate agar plates at 37°C in 5% CO2. Colonies are inoculated in Mueller-Hinton broth containing 0.25% glucose to reach an OD620 of 0.05 to 0.08 and incubated at 37°C with shaking until the OD620 reached 0.23 to 0.24. The bacteria were diluted in Geys balanced salt solution (Sigma) and 1% (wt/vol) BSA (assay buffer) at the working dilution of 104 CFU/ml. All mouse sera to be tested are heat inactivated for 30 min at 56°C. The total volume in each well is 50 µl, with 25 µl of serial twofold dilutions of test serum, 12.5 µl of bacteria at the working dilution, and 12.5 µl of baby rabbit complement. Positive serum samples are included in each assay. Controls include bacteria incubated with complement serum and immune sera incubated with bacteria and with complement inactivated by heating at 56°C for 30 min. Immediately after the addition of the baby rabbit complement, 10 µl of the controls are plated on Mueller-Hinton agar plates using the tilt method (time zero). The plate is incubated for 1 h at 37°C; 7 µl of each sample is spotted on Mueller-Hinton agar plates, and 10 µl of the controls are plated on Mueller-Hinton agar plates using the tilt method (time 1). The agar plates are incubated for 18 h at 37°C, and the colonies corresponding to time zero and time 1 are counted. Historically, the term complement referred to a heat-labile serum component that could lyse bacteria. Modern day immunology has recognized several other contributions of complement to host defenses as well. For example, complement can opsonize bacteria, cause phagocytosis, and regulate antibody responses. Complement can also have detrimental effects such as causing inflammation and tissue damage and triggering anaphylaxis. The above functions are termed the "complement cascade." More than 20 different serum proteins form the complement. These heat-labile proteins are produced by several cells including, hepatocytes, macrophages and gut epithelial cells. Some complement proteins act by binding to immunoglobulins or to membrane components of cells. Others are proenzymes which, upon activation, cleave some of the other complement proteins, yielding fragments that activate cells, increase vascular permeability or opsonize bacteria. The complement requires activation in order to function. Four pathways of activation of complement exist, including the classical pathway, the lectin pathway, the alternative pathway and the membrane attack (or lytic) pathway. Question 5 Aluminum hydroxide and MF59, in combination with CpG oligonucleotides, were the adjuvants used in the vaccine preparation. Adjuvants have the ability to potentiate the immune responses to an antigen and/or modify suitably the nature of immune responses. Hence, adjuvants are used to obtain strong and durable immune response to vaccine antigens (Brunner et al., 2010). Besides, the use of adjuvants in vaccine formulations leads to a reduced amount of the antigen required for successful immunization. The mode of action of aluminium-containing adjuvants such as aluminium hydroxide, a type B adjuvant, is to form a depot at the injection site which will increase the antigen concentration. The gradual and sustained release of the antigen from the depot enhances the interaction of the antigen with antigen presenting cells or APCs, resulting in increased antibody production (Brunner et al., 2010). The interaction between APCs and antigens occurs in an unspecific fashion and the results are due to an amplification of signal 1. Antigen recognition may be promoted through the specific stimulation of immune cells (Brunner et al., 2010). The other adjuvant used in this study MF59, also type B adjuvant, is an oil-in-water emulsion containing 5% squalene, 0.5% Tween 80 (a detergent) and 0.5% sorbitan trioleate. While its mechanism of action is yet to be fully understood, it is known that it does not restrain the distribution of the co-administered antigen by forming a depot at the injection site like aluminium hydroxide does (Brunner et al., 2010). Also, according to the same authors, MF59 reacts with APCs at the injection site and undergoes gradual dispersion to the lymph nodes where it concentrates and gets endocytosed by the lymph node- resident cells having characteristic APCs. Because MF59 can elicit a predominant Th1 cytokine response and cause efficient generation of cytotoxic T cells, it exhibits significant humoral as well as cellular potency. References Brunner R, Jensen-Jarolim E. and Pali-Scöll I., 2010. The ABC of clinical and experimental adjuvants – A brief overview. Immunology Letters 128: 29-35. Giuliani MM, Adu-Bobie J, Comanducci M, et al., 2006. A universal vaccine for serogroup B meningococcus. Proc Natl Acad Sci., 103 (29): 10834-10839. Read More