Elsevier

Vaccine

Volume 30, Issue 6, 1 February 2012, Pages 1038-1049
Vaccine

In vitro culture medium influences the vaccine efficacy of Mycobacterium bovis BCG

https://doi.org/10.1016/j.vaccine.2011.12.044Get rights and content

Abstract

The varied rates of protection induced by Mycobacterium bovis BCG vaccine against tuberculosis has been attributed to many factors such as genetic variability among BCG strains, rapid clearance of BCG in some populations, and different levels of previous exposure of vaccinated populations to environmental mycobacteria. However, the methods and conditions employed to prepare this vaccine for human usage by various manufacturers have not been investigated as potential factors contributing to the variation in vaccine efficacy. A review of the literature indicates discrepancies between the approach for growing BCG vaccine in the laboratory to assess immune responses and protective ability in animal models, and that employed for production of the vaccine for administration to humans. One of the major differences is in the growth medium used for routine propagation in the laboratory and the one used for bulk vaccine production by manufacturers. Here we compared the immunogenicity of the BCG vaccine grown in Middlebrook 7H9 medium, the most commonly used medium in laboratory studies, against that grown in Sauton medium, which is used for growing BCG by most manufacturers. Our results showed clear differences in the behavior of BCG grown in these different culture media. Compared to BCG grown in Middlebrook 7H9 medium, BCG grown in Sauton media was more persistent inside macrophages, more effective at inhibiting apoptosis of infected cells, induced stronger inflammatory responses and stimulated less effective immunity against aerosol challenge with a virulent Mtb strain. These findings suggested that the growth medium used for producing BCG vaccine is an important factor that deserves increased scrutiny in ongoing efforts to produce more consistently effective vaccines against Mtb.

Highlights

► We investigated whether growth media influence the vaccine efficacy of BCG. ► There were differences in immunogenicity for BCG grown in different media. ► Growth medium is an important factor affecting the efficacy of BCG vaccines.

Introduction

Mycobacterium bovis BCG is the only currently approved vaccine for prevention of tuberculosis (TB), and it continues to be used routinely in many regions with high levels of endemic TB. The original BCG strain was developed by Calmette and Guerin between 1908 and 1919 by carrying out 231 passages of virulent M. bovis in glycerinated bile potato medium [1]. This long-term serial cultivation led to spontaneous attenuation, resulting in a strain of M. bovis that was no longer virulent in healthy animals or humans and therefore was suitable for use as a live attenuated vaccine. However, the level of protection against pulmonary TB provided by BCG vaccination has been highly variable in different controlled trials around the world, ranging from 0 to 80% [2], [3], [4]. Three main hypotheses have been proposed to explain this variable efficacy. First, BCG has acquired additional mutations through many years of passage in culture which reduce its ability to stimulate protective immunity against Mycobacterium tuberculosis. Second, exposure to environmental mycobacteria alters the response to BCG and may lead to tolerance that blunts the vaccine effect. Third, there is a rapid clearance of BCG by vaccine recipients in some populations that does not provide sufficient exposure to the vaccine for development of long-term immunological memory [5].

Dissemination of the BCG vaccine over many years and geographic regions has led to the derivation of multiple substrains from the original strain, with as many as 49 production substrains being used at various times and places including the four major BCG vaccines in current use (BCG-Pasteur, -Danish, -Glaxo, and -Japan) [6], [7]. The multiplication of production sites has led to great variation in the culture conditions and the timing of the harvesting of BCG for vaccine production [8]. However, in most production laboratories that prepare BCG vaccines for administration to humans, the bacteria are grown as a pellicle on the surface of liquid Sauton medium [9]. In contrast, the liquid culture media employed to grow BCG for research purposes vary among different laboratories, with the most common medium used being Middlebrook 7H9. In fact, a review of the literature for the last 3 years led us to conclude that greater than 95% of published papers during this period evaluating immunogenicity and vaccine efficacy of BCG in vitro or in animal models have used organisms grown in Middlebrook 7H9 medium. Interestingly, it has been shown that BCG grown in Middlebrook 7H9 or Sauton media have different protein expression profiles, and different levels of sensitivity to reactive nitrogen intermediates [10]. Moreover, BCG vaccines grown in different culture media have been shown to induce distinct humoral immune responses in mice [11], and studies of other mycobacterial species have shown major effects of the growth medium composition on the secretion of virulence related proteins such as ESAT-6 and CFP-10 [12].

The above findings suggest that the growth medium used for preparation of BCG vaccines could have a significant impact on immunogenicity and vaccine efficacy, and that this may be an important factor contributing to variations in the properties of BCG strains both in laboratory studies and in clinical vaccine trials. In particular, it is striking that most BCG vaccines used for clinical trials or routine vaccination of humans have been grown in Sauton medium, whereas BCG used for laboratory based preclinical vaccine studies is usually grown in Middlebrook 7H9 medium. This discrepancy between methods used in the laboratory versus the clinical setting prompted us to investigate whether these two different growth conditions may have any influence on immunological properties and protective efficacy of the BCG vaccine in the mouse model. Our results show that this is indeed the case, and indicate that growth conditions prior to in vivo inoculation of BCG have a significant effect on the subsequent immune response in the vaccinated host.

Section snippets

Mice

Six- to 8-week-old female wild type (C57BL/6) and severe combined immunodeficiency (SCID; also C57BL/6 background) mice were obtained from Jackson Laboratories (Bar Harbor, Maine). All mice were maintained in specific pathogen-free conditions, and were transferred to biosafety level 3 conditions for infection with M. tuberculosis. All procedures involving the use of animals were in compliance with protocols approved by the Albert Einstein College of Medicine Institutional Animal Use and

Increased resistance to killing in vitro and in vivo of BCG grown in Sauton medium

In order to assess the influence of growth conditions on the intracellular survival of BCG, we infected BMM with BCG organisms (Pasteur strain) grown in either Sauton (BCG-S) or M7H9 (BCG-M) media, and analyzed the numbers of viable bacilli (CFUs) at different time points. We observed a significantly higher survival of BCG-S compared to BCG-M, starting from 24 h through 7 days post infection (Fig. 1A). This effect was observed with three different substrains of BCG (Pasteur, Danish and Tice

Discussion

Despite the fact that the BCG vaccine has been administered to people for nearly a century, the factors influencing its protective efficacy against Mtb infection remain poorly understood. In the current study, we have examined the potential impact of the growth medium used to propagate BCG on its subsequent behavior in infected cells and animals. We were motivated to carry out this study by the observation that many vaccine producers have used and continue to use Sauton medium for growth of

Acknowledgements

This work was supported by NIH/NIAID grants PO1AI063537 (SAP, WRJ and JC) and 1RO1AI093649 (SAP). MG was supported by NIH Training Grant 5T32CA009173. Flow cytometry studies were carried out using core facilities supported by the Einstein Cancer Center (NIH/NCI CA013330) and the Einstein Center for AIDS Research (NIH AI-51519).

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    1

    These authors contributed equally to this work.

    2

    Present address: Grupo de Immunologia Celular e Inmunogenetica, Universidad de Antioquia, Medellin, Colombia.

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