Production and control of a Brazilian tailor-made meningococcal B vaccine

Meningococcal disease has been a public health problem in Brazil since the serogroups A and C epidemics occurred in the 1970s. The Oswaldo Cruz Foundation in Brazil has been working to develop a serogroup B meningococcal vaccine composed of detergent-treated outer membrane vesicles (OMV) and detoxified endotoxin (dLOS) from Neisseria meningitidis serogroup B prevalent strains. Experimental vaccine were produced in a pilot-scale under Good Manufacturing Practice condition (GMP). Physicochemical and biological controls were established based on what has been reported previously for OMV vaccines. The developed vaccine contained the main class 1, 2, 3, and 5 proteins, some minor iron-regulated proteins, and 5–10% residual lipopolysaccharide related to total protein content. The dLOS was added, as a vaccine component, in half of the total protein amount. The pyrogenicity of the final products, based on the residual LOS in OMVs, varied from 1.01 to 2.24 ng LOS/kg rabbit. Three experimental vaccines, were highly immunogenic in mice with better performance using higher antigen concentrations.


INTRODUCTION
Meningococcal disease remains a worldwide health problem and has been a public health 39 problem in Brazil since the groups A and C epidemics of the 1970s. Serogroup B caused 80% 40 of the meningococcal disease cases throughout the 1980s with a significant number of cases in 41 children < 2-years-of-age [1,2]. 149 The bacterial strains, was streaked on Columbia blood agar (Merck 1.10455.0500), plated with 150 5% horse blood (CBA), and incubated overnight at 37°C in 5% CO 2 . The next morning, 10-20 151 colonies, were subculture on another CBA plate and incubated for 4 h at 37°C in 5% CO 2 . After  The experimental vaccines were tested by intraperitoneal injection of one human dose, but not 170 more than 1 mL of experimental vaccine into 5 mice (weight, 17-22 g) diluted in diluent. Two 171 guinea pigs (weight, 250-350 g) were also injected (i.p.) with the equivalent of 10 human doses in a volume  5 mL. The  The protein profile of the OMV preparations, before vaccine formulation,was analyzed by 12%

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The experimental formulations were sterile, more than 80% of vaccine antigens adsorbed in Al 274 (OH) 3 , had appropriate pH, sialic acid, aluminum, and antigen concentrations in accordance 275 with the established quantitative limits. The OMVs in residual LOS varied from 5 to 10% of 276 the OMV protein (Table 1).

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The three experimental vaccines induced significant increases in total IgG (p < 0.001) in 303 response to the OMVs (    In addition, the bactericidal titers were significantly higher for N44/89 than what was observed 323 to N603/95 after the third dose (Table 3). Both mice and guinea pigs survived to 7 days of time observation without any sign of toxicity, 337 such weight loss, due to experimental preparations and reference vaccines administration.

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According to the WHO requirements for polysaccharide vaccines against N. meningitidis, the 339 final product should not induce fever in rabbits at a 1:4,000 dilution of the human dose.  (Table 4). The iron starvation strategy for vaccine strain growth induced IRP expression in OMV outer 356 membrane from both vaccine strains in the molecular weight range between 60 and 90kDa.

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Por A and other class proteins were well expressed in OMV from N44/89 and N603/95 ( Figure   358 3).  Table 5 shows the percentages of expression of the main OMV proteins of the vaccine strains 365 used as reproducibility parameter of the production process of this vaccine component.  The structural changes in Lipid A from raw LOS after the NaOH treatment are shown in the 370 SDS profiles. Both molecules had two bands but the molecular weights of dLOS were lower 371 than those shown in LOS profile before alkali detoxification (Figure 4).  (Table 1),. The rabbit pyrogen test has been used historically to measure pyrogenicity potential of human 464 and animal vaccines. However, whole cell and OMV based vaccines, which intrinsically 465 contain relatively high concentration of pyrogenic material, has been a source of great concern.
466 Several studies have been reported to define the best biological tests that can be used as safety