Biomedical Engineering Reference
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Figure 3.6. Immunoblot showing binding specificity of EBA-175 RII and RII-NG proteins.
Recombinant region II was allowed to bind RBC or neuraminidase-treated RBC that had sialic
acids removed. The mixture was spun through oil-separating RBC with bound or unbound RII or
RII-NG protein. The RBC bound RII or RII-NG protein was eluted off RBC with high salt buffer.
Recombinant RII or RII-NG bound normal RBC but not neuraminidase-treated RBC. The immu-
noblot was probed with rabbit anti-RII antibodies.
sialic-acid-dependent binding to human erythrocytes (Fig. 3.6). Therefore, the CQA
identified as maintaining functional activity was unchanged.
Structural Comparisonof EBA-175RII andRII-NG. PurifiedEBA-175RII-NG
protein was used for crystallographic studies and shown to crystallize as a dimer [42]. For
the purpose of this discussion, a space-filling representation of the dimeric structure is
shown in Fig. 3.5. Four of the fivemutations are shown in red and can be observed together
whenviewed as a dimer. The fifthmutation is near the amino terminus of the recombinant
protein and is not represented in the crystal structure. No significant changes were
observed due to the introduction of the amino acid substitutions.
Comparison of EBA-175 RII and RII-NG Immunogenicity and Parasite
Growth Inhibition. To evaluate the immunogenicity of EBA-175 RII and RII-NG,
rabbits were immunized with both recombinant proteins [33]. The results of the ELISA
titers as detected by a homologous and heterologous capture antigen are shown in
Table 3.3. No marked differences were observed by ELISA. Rabbit immunoglobulin G
was purified from the immune sera and tested in a P. falciparum growth inhibition assay,
which generally measures the capacity of antibodies to interfere with erythrocyte
invasion. The level of growth inhibition appeared similar for the two different recombi-
nant forms of EBA-175 when measured against two different strains of P. falciparum,
which may use different erythrocyte invasion pathways [16]. Again, another important
CQA appeared to be unchanged.
Human Clinical Testing. EBA-175 RII-NG has been cGMP manufactured for
human clinical testing by a contract facility. The recombinant RII-NG protein is for-
mulated on aluminum phosphate [43] and is currently being tested in a phase I dose
escalation safety study. The results of this phase I human trial may be available in 2009.
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