Submitted to: Vaccine
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2010
Publication Date: 5/7/2010
Citation: Noh, S.M., Zhuang, Y., Futse, J.E., Brown, W.C., Brayton, K.A., Palmer, G.H. 2010. The immunization-induced antibody response to the Anaplasma marginale major surface protein 2 and its association with protective immunity. Vaccine. 28:3741-3747.
Interpretive Summary: Many vector borne pathogens, including Anaplasma sp. and Plasmodium sp. and Borrelia sp., evade the immune system by rapid variation in surface expressed proteins, known as antigenic variation. These proteins generally incite a strong antibody response which then acts to clear organisms displaying that particular protein variant from circulation. Subsequently, organisms expressing a new variant, to which antibody does not yet exist, replicate and become responsible for another wave of parasitemia or bacteremia. In the case of Anaplasma marginale, a tick transmitted disease of cattle which causes anemia, major surface protein 2 (Msp2) is one of the primary proteins responsible for antigenic variation and escape from immune clearance. Msp2 is expressed from a single site in the genome of A. marginale. This expressed protein has a central highly variable region, which is flanked by conserved sequences. The variability in the central region (termed the hypervariable region) is derived from the recombination of gene cassettes (termed pseudogenes) into the single expression site. There are multiple pseudogenes scattered throughout the genome. Early in infection, the hypervariable region within the expression site will be derived from single, whole, or parts of two pseudogenes, termed simple variants. During persistent infection, the hypervariable region will become more complex as short cassettes from multiple pseudogenes recombine in to the expression site. The expressed variants derived from three or more pseudogenes are termed complex variants. As the immune system exerts selective pressure on the simple variants, they are cleared from circulation, and complex variants will predominate. Animals naturally infected with A. marginale, given time, are able to control but not clear the infection. This is in contrast to immunized animals, in which 40 to 70% are protected from infection, while nearly all immunized animals are protected from high levels of bacteremia. In order to identify the essential components of an effective vaccine, we pose the question: What is the difference in the immune response between naturally infected and immunized animals? Because Msp2 is responsible for successful evasion of the immune system in naturally infected animals and high levels of antibody correlate with protection in immunized animals, we focused on the immune response to Msp2 in controlling and preventing infection in immunized animals. The first goal of these experiments was to determine if immunization induced anti-Msp2 antibody targeting different areas of Msp2 than that induced during infection; specifically whether the antibody response was shifted toward recognition of the conserved region in immunized animals as compared to infected animals. This immunity against the conserved regions could prevent immune escape of new variants and result in the clearance observed following immunization but not during infection. The second goal was to determine if the anti-Msp2 immune response induced by immunization resulted in selection for complex HVR variants, suggesting the immune response targeting Msp2 was at least partially responsible for the prevention of high levels of bacteremia. To address these questions, animals were immunized with purified outer membrane proteins, linked protein complexes, or adjuvant and challenged with A. marginale. The two immunized groups of animals were similarly protected. There were no differences in the anti-Msp2 antibody repertoire among all groups of animals. Thus, we conclude that a shift in anti-Msp2 antibody repertoire does not contribute to immunization-induced protective immunity. The majority of the Msp2 expressed variants was of the simple type in the groups that received only adjuvant or were immunized with outer membranes, suggesting that the selective pressure of the anti-Msp2 immune r
Technical Abstract: Many vector-borne pathogens evade clearance via rapid variation in immunogenic surface expressed proteins. In the case of A. marginale, the generation of major surface protein 2 (Msp2) variants allows for immune escape and long-term pathogen persistence. In the experiments reported here, we pose two questions in order to examine the possible roles the anti-Msp2 immune response may play in immunization-induced immunity; 1) Does immunization alter the anti-Msp2 antibody repertoire? 2) Does the anti-Msp2 immune response exert selective pressure on Msp2 and thus help control the initial bacteremia? To address these questions, animals were immunized with purified outer membrane proteins, cross-linked protein complexes, or adjuvant and challenged with A. marginale. The immunized groups of animals were similarly protected. After immunization, the antibody repertoire targeting the conserved and hypervariable regions of Msp2 was measured using enzyme-linked immunosorbent assays. After challenge, the complexity of the Msp2 hypervariable region in expressed variants was determined by cloning and sequencing the Msp2 expression site. The immunized groups of animals were similarly protected from challenge. There were no differences in the anti-Msp2 antibody repertoire among all groups of animals. Thus, we conclude that a shift in epitope recognition of the anti-Msp2 antibody does not contribute to immunization-induced protective immunity. The majority of the Msp2 expressed variants was of the simple type in the groups that received only adjuvant or were immunized with outer membranes, suggesting that the selective pressure of the anti-Msp2 immune response was insufficient to drive Msp2 expression toward complex variants, and thus may have little effect on the initial control of bacteremia. In the group immunized with protein complexes, a majority of the Msp2 expressed variants were of the complex type suggesting immunogen-dependent mechanism of immune control may occur in similarly protected animals.