G-Protein-Coupled Receptors (GPCR) and Vaccine Development Module
I. IntroductionIn this Vaccine Module, we presents how G-protein-coupled receptor (GPCR) could potentially play a role in the development of new vaccines, as shown in studies on our organism of interest, Rhipicephalus microplus, formerly known as Boophilus microplus. With its associated agricultural problems, this parasitic tick has a significant economic impact on a variety of livestock species.
II. Rhipicephalus microplusThe common name for this species is either the “cattle ticks” or “southern cattle ticks.” They are small arachnids of the order Ixodida in the same subclass as mites. Ticks are ectoparasites (i.e., external parasites) living by hematophagy, feeding on the blood of mammals, and are known vectors of a number of diseases including Lyme Disease, Q Fever, Rocky Mountain Spotted Fever, tularemia, and bovine anaplasmosis. A vector is any agent (person, animal, or microorganism) that carries and transmits an infectious pathogen into another living organism [1,2,3,5,17].
The cattle tick has been found on organisms ranging from cattle to buffalo, horses, donkeys, goats, sheep, deer, pigs, dogs, and some other wild animals, most commonly in Asia, parts of Australia, Madagascar, Southeastern Africa, the Caribbean, South and Central America, and Mexico. Although the tick has been eradicated in the US [2,3,17], there are still sporadic occurrences in the buffer zone along the US-Mexican border. Cattle fever ticks have expanded beyond the quarantine zone in South Texas. The problem is that there are currently no vaccines approved for use within the US for controlling these vectors. Figure 1: World-wide distribution of the Cattle Tick, Rhipicephalus microplusR. microplus is considered to be the most important tick parasite of livestocks worldwide. Burdens of heavy tick infestations on animals can decrease production and damage cattle hides. R. microplus can also transmit protozoal parasites Babesia bigeminaand and Babesia bovis, causing babesiosis, and Anaplasma marginale leading to anaplasmosis [2,3]. Figure 2: The Cattle Tick, Rhipicephalus microplusIt is pertinent to study the life cycle of this tick in order to understand the mechanism of parasitism. R. microplus is a one-host tick with all stages spent on one animal. After hatching of the eggs, the larvae crawl up grasses or other plants to find their potential hosts, but they may also be blown by the wind. In the summer, the tick can survive for as long as 3 to 4 months without feeding. In cooler temperatures, they may live without food for up to 6 months. Ticks that do not find their host will eventually die of starvation. Newly attached seed ticks (i.e., larvae) are usually found on the softer skin inside the thigh, flanks, and forelegs; they may also be seen on the abdomen. After feeding, the larvae molt twice and become nymphs before turning into adults. The feeding takes place only once in each developmental stage (larva, nymph and adult) but over several days [17]. Adult male ticks become sexually mature after feeding, and mate with adult feeding females. A mated female tick detaches from the host and deposits a single batch of many eggs in the surrounding. These eggs are typically placed in crevices, debris, or under stones. The female tick dies after ovipositing. Ticks in the subgenus Boophilus have a life cycle that can be completed in 3 to 4 weeks; this characteristic can result in a heavy tick burden on animals [2,3,17]. Figure 3 below illustrates this cycle Figure 3: Life cycle of the Cattle Tick, Rhipicephalus microplusIn arthropod research, there is an absence of a complete Chelicerate genome, which includes ticks, mites, spiders, scorpions and crustaceans. Model arthropod genomes such as Drosophila and Anopheles are too taxonomically distant for a reference in tick genomic sequence analysis. The genome of R. microplus has an estimated size of 7.1 Gb, a very small one comparing to the human genome of over 3 billion bp. NCBI has a published Genome Assembly/Annotation report that can be found directly at http://www.ncbi.nlm.nih.gov/genome/?term=rhipicephalus%20microplus.
III. VaccinesTo understand the goal of vaccine research, we need to know vaccine development and its history; vaccines are common biological preparations developed to improve immunity against particular diseases. A vaccine is typically made with a weakened or inactivated form of the disease-causing microbe, its surface proteins, or toxins. The goal of vaccination is to stimulate the immune system so that the body can recognize the vaccine as a foreign agent and develop an ability to neutralize the actual live microbe in future encounters [9,14,16]. Vaccines do not guarantee complete protection from a certain disease. For example, the host's immune system may not respond adequately or its immunity could have been lowered due to the lack of B-cells capable of constructing an antibody for that particular antigen [14]. Even if the host develops antibodies, its immune system may not be perfect. If re-infected, the immune system may not be able to defeat the foreign agent immediately, and in this case, however, the infection would be less severe than normal and would heal faster [9]. A. Vaccine PerformanceVaccine Efficacy is defined as the reduction in the incidence of a disease by comparing vaccinated and unvaccinated people. The is usually measured during phase I or II of clinical trials by giving one group a vaccine and comparing the incidence of disease in that group to another group of people who did not receive the vaccine. Ideally, these would be double-blind, randomized, and controlled trials [15,18,19,20]. The equation is
Advantages: A vaccine efficacy study includes rigorous control for biases afforded by randomization, prospective, active monitoring for disease attack rates, and careful tracking of vaccination status; often there is, at least for a subset of the study population, laboratory confirmation of the infectious outcome of interest and a sampling of vaccine immunogenicity. Disadvantages: The complexity and expense of performing them, especially for relatively uncommon infectious outcomes for which the sample size required is driven upwards to achieve clinically useful statistical power. Ultimately, vaccine efficacy studies measure outcomes beyond disease attack rates (i.e., proportion of subjects who become sick from exposure to the vaccine or not), including hospitalizations, medical visits, and costs. The external validity of the results of a vaccine efficacy study to a larger, non-study population may be lowered by differences between the study cohort and the population as a whole [15]. Vaccine Effectiveness is, often confused with vaccine efficacy, defined as a “real world” view of how a vaccine reduces disease in a population, despite having a high vaccine efficacy in prior well-controlled clinical trials. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under more natural field conditions rather than those controlled conditions in a clinical trial. Vaccine effectiveness is proportional to vaccine potency (i.e., vaccine efficacy) but is also affected by how well target groups in the population are immunized [4,10]. The outcome data (i.e., vaccine effectiveness) are expressed as a rate difference, with use of the odds ratio (OR) for developing infection despite vaccination: Effectiveness = (1 – OR) x 100. B. Vaccine Efficacy Comparisons Based on the Type of Study
Table 1: Vaccine efficacy comparison table based on particular methods of studyC. Vaccine Efficacy Study Vocabulary
D. Types of VaccinesThe most common types of vaccinations represent different strategies used to reduce the risk of illness or infection, while having the ability to induce an immune response [9,16].
E. The Cattle Tick VaccineBabesiosis or “cattle fever” was eradicated from the United States, from 1906 to 1943, by eliminating its vectors R. microplus and Rhipicephalus annulatus (a similar tick species). Before its eradication, babesiosis costed the US an estimated $130.5 million in direct and indirect annual losses; in current dollars, the equivalent would be $3 billion [5]. R. microplus and R. annulatus still exist in Mexico, and a permanent quarantine zone is maintained along the US-Mexican border to prevent their reintroduction into the US. Within this zone, the USDA’s Animal and Plant Health Inspection Service (APHIS) conducts a surveillance program to identify and treat animals infested with these exotic ticks. Recently, increased numbers of infestations have been recorded in the quarantine zone [5]
Figure 4: Heavy infestation of cattle ticksControl of cattle tick infestations has been primarily by application of acaricides, which has resulted in environmental pollution and the unwanted selection of resistant ticks. Arcaricides are pesticides used to kill ticks and mites [5], used commonly in agriculture with very high toxicity. “Commercial tick vaccines for cattle based on the R. microplus Bm86 gut antigen have proven to be a feasible tick control method that offers a cost-effective, environmentally friendly alternative to the use of acaricides" [1]. The current problem is that cattle fever ticks have expanded beyond the quarantine zone in southern Texas. While Bm86 vaccine has been developed in Australia and Cuba, there are no vaccines approved for use within the US for controlling these vectors. The Bm86 vaccine is a glycoprotein, located predominantly on the surface of tick mid-gut digestion cells, without known functions [1,5]. As a sidenote, glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a co- or post-translational modification. Glycoproteins are often important integral membrane proteins, where they play a role in cell–cell interactions. Glycoproteins are also formed in the cytosol, but their functions and the pathways producing these modifications in this compartment are less well-understood [6]. Bm86 is a recombinant antigen identified through a complex series of protein fractionations followed by vaccination trials in cattle to assess the antigenic efficacy against R. microplus [1]. The Bm86 vaccine has variable efficacy against cattle fever ticks. A possible explanation for this variation in vaccine efficacy is amino acid sequence divergence between the recombinant Bm86 vaccine component and native Bm86 expressed in ticks from different geographical regions. Commercially available vaccines against cattle fever ticks, which are approved for use outside of the US, including Gavac® (Heber Biotec; Havana, Cuba), TickGARD (Hoechst Animal Health; Australia), and TickGARDPLUS (Intervet Australia; Australia), are based on the recombinant form of the concealed midgut antigen, Bm86 [1,5].
Figure 5: Commercially available vaccines against cattle fever ticksCommercial tick vaccines reduced tick infestations on cattle and the intensity of acaricide usage, as well as increasing animal production and reducing transmission of some tick-borne pathogens. Commercialization of tick vaccines has been difficult owing to previous constraints of antigen discovery, the expense of testing vaccines in cattle, and company restructuring, the success of these vaccines over the past decade has clearly demonstrated their potential as an improved method of tick control for cattle [1]. Development of improved vaccines in the future will be greatly enhanced by new and efficient molecular technologies for antigen discovery and the urgent need for a tick control method to reduce or replace the use of acaricides, especially in regions where extensive tick resistance has occurred. F. Vaccines and GPCRGPCR is one of the most commonly used and successful targets for drugs with approximately 40-50% of all modern medicines interact with this protein group. GPCRs are prime targets of interest due to their physiological relevance. GPCR’s role as contributor to the information flow into cells also makes that they are associated with a multitude of diseases. The prominent role of GPCR in drug development will most likely increase in the future [8].
IV. G-Protein Coupled Receptors (GPCRs)GPCR is also known as 7-transmembrane protein (7TM) receptors, heptahelical receptors, serpentine receptors, or G-protein-linked receptors, which is a very large family of protein receptors. The main function of GPCRs is to sense stimuli from outside of the cell and activate signal transduction pathways inside the cell and ultimately to produce the biological responses. GPCRs are considered transmembrane receptors because they pass entirely through the cell membrane. They are called 7-transmembrane receptors because the proteins pass through the cell membrane exactly 7 times [6,7,8,11,12,13].
Figure 6: GPCR and intracellular responses to stimuliAlthough GPCRs are the largest and most diverse group of membrane receptors, they are found only in eukaryotes, which are organisms with cells containing complex structures enclosed within membranes [6,12]. GPCRs are located on the cell surface, which is a prime location for receiving a variety of signals in the form of light energy, peptides, lipids, sugars, neurotransmitters, or proteins (the red dot in Figure 7). GPCRs have a wide variety of functions not limited to stimulus-response pathways and have also been linked to a wide array of biological and pathological conditions; they are thus very common drug targets [7,8,12]. Current research challenges include understanding GPCR regulation related to both normal and abnormal cellular processes. Figure 7: The 7 transmembrane proteins of GPCR and the stimulus (red dot)A. GPCR Vocabulary
B. GPCR StructureGPCRs are integral membrane proteins and they possess 7 membrane spanning domains (as can be deduced from their name and seen in Figure 7 above). Most GPCRs share a similar architecture and structure (conserved through evolution). The GPCR itself consists of a single polypeptide folded into a globular shape, and embedded into a cellular membrane. There are segments of the polypeptide loop inside and outside of the cell. Also, extracellular loops form part of the pocket, where signaling molecules bind to GPCRs [7,11,13]. C. Physiological Roles of GPCRsInitially, GPCRs interact with G-proteins in the plasma membrane; they are specialized proteins with the ability to bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP) [13]. The G-proteins that interact with GPCRs are heterotrimeric, meaning the protein has three different subunits (alpha-, beta-, and gamma-subunit). Alpha- and gamma-subunits are attached to the plasma membrane of the cell through lipid anchors. Then, external signaling molecules bind to a GPCR, causing conformational changes, triggering the interaction between the GPCR and the G-protein [11]. The mechanisms, or steps of GPCR in relation to their physiological roles include first ligand binding, then conformational changes in the structure, activation or deactivation, signaling and regulation. Ligand binding is the first step in a signal transduction pathway, when signaling molecule activates a cell surface receptor. The receptor then undergoes conformational changes, involving disruption of a strong ionic interaction between the 3rd and 6th transmembrane helices. The next step is G-coupled protein activation or deactivation. The conformational changes typically facilitate activation of the G-protein heterotrimer. Depending on the type of G-protein to which the receptor is coupled, a wide variety of downstream signaling pathways can be activated (based on highly specific interactions). GPCR Signaling can be independent or dependent. Then the receptor is regulated [6,7,8,11,12,13]. D. Classification of GPCRsGPCRs are a superfamily of proteins (how many exist is unknown). The superfamily is divided into 3 main classes (A, B, and C) plus 3 other classes (D, E, and F) that are rare and unique. Despite the lack of detectable sequence similarity among the classes, the structure and mechanisms of signal transduction are similar across all GPCRs. It is interesting that the human genome encodes thousands of GPCRs. Also, there are several web-servers and bioinformatics tools available to predict GPCR sites and their classifications based on their amino acid sequence [6,8]. GPCR Class A (Rhodopsin-like) receptors are the largest class of GPCR genes (accounts for nearly 85%). This group can be subdivided into 19 more specific groups. This is a widespread protein family which includes hormones, neurotransmitters, and light receptors. These molecules all allow for extracellular signaling through their interaction with G-proteins. GPCR Class B (Secretin Receptor Family) receptors are typically regulated by peptide hormones from the glucagon hormone family. Three subfamilies have been recognized in this class. GPCR Class C (Metabotropic Glutamate/Phermone) receptors are a type of glutamate receptor that is activated indirectly through certain metabotropic processes. Glutamate receptors always bind the amino acid glutamate. This amino acid functions as an excitatory neurotransmitter. GPCR Class D (Fungal Mating Pheromone Receptors) are involved in the response to mating factors on the cell membrane. Two specific receptors are STE2 and STE3. The amino acid sequences for both receptors contain high amounts of hydrophobic residues grouped into seven domains (believed to be very similar to the structure of GPCRs). GPCR Class E (Cylic AMP Receptors) usually occur in slime molds. These receptors coordinate the aggregation of individual cells into a multicellular organism. They also regulate the expression of developmental genes. GPCR Class F (Frizzled/Smoothened) refers to a set of GPCRs serving as receptors in many signaling pathways. Frizzled proteins play a key role in determining cell polarity, embryonic development, neural synapses, cell proliferation, and many other processes. This differs from smoothened (SMO) because these proteins are the molecular target for teratogen cyclopamine encoded by the SMO gene. E. GPCR Prediction ServersThere are several web-servers and bioinformatics tools available to predict GPCR sites and their classifications according to their amino acid sequences. 2. GPCR HMM http://gpcrhmm.sbc.su.se/ 3. GPCR-GIA http://peds.oxfordjournals.org/content/22/11/699.long 4. GPCRpred http://www.imtech.res.in/raghava/gpcrpred/ 5. PCA-GPCR http://www1.spms.ntu.edu.sg/~chenxin/PCA_GPCR/index.html
V. Rhipicephalus microplus, Vaccinations, GPCR, and Their RelatednessRhipicephalus microplus is an economically important tick that parasitises a variety of livestock species. Control of cattle tick infestations has been primarily by application of acaricides, which has resulted in selection of resistant ticks and environmental pollution. In other countries, the success of vaccines over the past decade has clearly demonstrated their potential as an improved method of tick control for cattle. Currently, there are no vaccines approved for use within the United States for controlling R. microplus. Development of improved vaccines in the future will be greatly enhanced by new and efficient molecular technologies for antigen discovery and the urgent need for a tick control method to reduce or replace the use of acaricides, especially in regions where extensive tick resistance has occurred. One of the most commonly used and successful targets for drugs is a specific protein group called G-Protein Coupled Receptors (GPCRs). Approximately forty to fifty percent of all modern medicines interact with this protein group. GPCRs are prime targets of interest due to their physiological relevance. GPCR’s role as contributor to the information flow into cells also makes that they are associated with a multitude of diseases. The prominent role of GPCR in drug development, will most likely increase in the future.
VI. References[1] Canales, M., Almazan, C., Naranjo, V., Jongejan, F., and de la Fuente, J. (2009) Vaccination with recombinant Boophilus annulatus Bm86 ortholog protein, Ba86, protects cattle against B. annulatus and B. microplus infestations. BMC Biotechnology. 9:29. http://www.biomedcentral.com/1472-6750/9/29. Accessed September 4, 2014. |