and B. pseudomallei
are two highly pathogenic species of bacteria to human. The mortality rate of patients with melioidosis and glanders following B. pseudomallei
and B. mallei
infections remains very high, despite aggressive antibiotic treatments. The mechanisms of host-pathogen interaction for Burkholderia
bacteria are evidently unique. The immunity developed from the disease or repeated exposure to B. pseudomallei
seemed insufficient to prevent a relapse or to provoke a protective response for primary infection (3
). Development of various approaches of immunotherapy has been attempted (2
). Adoptive transfer of mononuclear leukocytes from B. pseudomallei
-immunized mice was unable to provide protection against a lethal challenge of the bacteria (1
). On the other hand, B. mallei
- and B. pseudomallei
-specific MAbs were reported to be effective in protecting the animals against lethal challenges of the bacteria (13
). Our laboratory has developed more than 100 MAbs against Burkholderia
bacteria. In an effort to search for potent therapeutic MAbs, we first examined the in vitro
bactericidal effects of these MAbs. It has shown that MAbs alone did not affect bacterial growth; however, in the presence of complement and phagocytes, a number of the selected MAbs possessed strong bactericidal effects. The differentiated HL-60 phagocytes have been widely used by many researchers in different opsonic assays (10
). However, variations in the opsonic bactericidal activities among experiments did occur occasionally in our study. Multiple repeats for these experiments were employed to reduce the range of standard deviations.
It is important to note that normal serum (from either human, rabbit, or guinea pig) per se
added at a higher concentration could produce a significant inhibitory effect on the bacteria, especially on B. mallei
. This kind of bactericidal effect had been suggested as the result of complement and low-binding-affinity, broad-spectrum natural antibodies present in these sera (14
). Heat inactivation of the sera at 56°C for 30 min effectively abolished the bactericidal effect of these sera, indicating that complement was involved. In our present study, we selected guinea pig serum as the source of complement. The serum sample was first diluted 20-fold before being added to the mixture of opsonic assay solution. The final concentration of guinea pig serum in the assay was 0.5%. At this concentration, the guinea pig serum alone had no inhibitory effect on the growth of Burkholderia
bacteria, but it provided sufficient complement to mediate the bactericidal effects of MAbs in the presence of phagocytes.
Our previous study revealed that most MAb groups are able to recognize more than one species among the Burkholderia
genus, except for the group C MAbs, which recognize only B. mallei
). The present study showed that the opsonic bactericidal activities varied significantly not only among different groups of MAbs but also among MAbs in the same group. This significant variation of antibacterial effects among MAbs in the same antibody group is most likely due to differences in the locations of their respective epitopes present on the same antigens and in their antigen-binding affinities. A surface plasmon resonance (SPR) biosensor (Biacore) is presently employed to study the binding interactions between these MAbs and their respective antigens to evaluate these two possibilities (18
). All MAbs found to possess strong opsonic bactericidal activities were in the groups of B, C, and E of the initial classification. Our ELISA demonstrated that the location of these antigens/epitopes played an essential role in determining the effectiveness of the opsonic activity of the MAbs. Capsular PS and LPS on intact bacteria are apparently more accessible to the MAbs than the proteins and glycoproteins. The accessibility of the target antigens on intact bacteria to the MAbs should be prerequisite to antibody-mediated phagocytosis. As a result, MAbs against intracellular antigens are not suitable for antibody-mediated phagocytosis.
It is interesting to note that antisera from the mice hyperimmunized with heat-killed B. mallei and B. pseudomallei, in comparison with many B. pseudomallei- and B. mallei-specific MAbs, produced only weak opsonic antibacterial effects in our assay. One possibility could be that the amount of anti-capsular PS and LPS antibodies in the serum samples are too low to offer strong bactericidal effects under our assay condition since antisera also contain numerous antibodies against glycoproteins/proteins.
Isotyping of the MAbs reveals another interesting finding: the isotypes of the MAbs do not affect their opsonic activity. There are two types of receptors present on the phagocytes that mediate opsonophagocytosis. One is the Fc receptor, and the other one is the complement receptor. HL-60 cells have previously been shown to express both receptors after induction to differentiate (10
). As discussed earlier, under our experimental condition, the concentration of complements was too low to opsonize the B. mallei
or B. pseudomallei
bacteria. MAbs of various isotypes alone activated opsonophagocytosis only moderately. It is in accordance with the fact that Fc receptors, such as FcγR II on the HL-60 cells, are low-affinity receptors for IgG1, IgG2, and IgG3 (10
). However, in the presence of both, complement and antibodies synergistically opsonized the bacteria, leading to a much greater extent of phagocytosis by the HL-60 cells. Therefore, binding of the MAb onto bacteria can further fix the complements onto the antibody-bacteria immune complex, resulting in a much stronger binding to the HL-60 cells and a more effective elimination of the bacteria.
Our in vivo
protective study showed consistent results to our in vitro
bactericidal study, which indicates that the opsonic bactericidal assay can provide valuable information in determining the effectiveness of the MAbs to confer passive immune protection against intranasal B. pseudomallei
and B. mallei
challenge. Both capsular PS and LPS were known to be important virulence factors in bacterial infection. Trevino et al. have shown that anti-LPS MAbs are effective in protecting mice from lethal aerosol challenge of B. mallei
). Our studies further showed that in addition to anti-LPS MAbs, anti-capsular PS MAbs were even more effective in early protection against intranasal challenge of B. mallei
. Previous studies by Jones et al. have shown that a cocktail of mixed MAbs against exopolysaccharide, LPS, and proteins demonstrated a significant protective effect against intraperitoneal challenge of B. pseudomallei
). Compared individually, both anti-exopolysaccharide and anti-LPS MAbs performed better in protection than the anti-protein MAbs. Our studies are in agreement with their study and further showed that the location of the antigens plays an important role in determining the effectiveness of the antibodies. Our studies also showed that not all anti-capsular PS or LPS MAbs are equally effective. As mentioned earlier, the location of each epitope and the affinity between the antibodies and their respective epitopes may affect their ability in performing opsonophagocytosis and complement fixation. It is known that the aerosol route can induce a much more acute and severe infection than the intraperitoneal route and therefore is more difficult to combat. Our MAbs were shown to provide protection against intranasal challenge of B. pseudomallei
for at least 3 weeks and B. mallei
for 2 weeks. This pioneer study suggests that our model may be suitable for searching MAbs with high efficiency in combating B. pseudomallei
and B. mallei
. A more comprehensive and thorough animal study will ensue to show the effectiveness of these MAbs in stopping or deterring melioidosis and glanders in the long run. Furthermore, these MAbs with high performance in anti-B. pseudomallei
and anti-B. mallei
activities could potentially be genetically modified and developed into effective therapeutics in prevention or treatment against infections by these two highly pathogenic bacteria in human.
In conclusion, we have examined bactericidal activity of a large panel of MAbs specific to various antigens of B. pseudomallei and B. mallei by in vitro opsonic assay and found that MAbs with strong opsonic activities are those with specificity against either the capsular PS or LPS of the bacteria. Several MAbs were selected to be evaluated for their effects on passive protection against Burkholderia infection in BALB/c mice by intranasal challenge with a lethal dose of bacteria. It was again found that the MAbs with the highest protective efficacy are those reactive to either the capsular PS or LPS.