Borrelia antigens

The antigens of Borrelia burgdorferi can be separated according to their molecular weight by means of SDS-Page (laboratory method for the separation of proteins) and determined by a size marker. About 853 genes (!) are responsible for the immunogenicity of the Borrelia burgdorferi and for the complex immune response of the host.

Immunity and pathogenicity of Borrelia burgdorferi: Borrelia burgdorferi has the ability of adhesion, invasion, persistence and immune evasion. These abilities serve to establish, multiply and spread the pathogen in the infected organism. During infection, borrelia migrate into various tissues of the host organism in order to enter so-called "immune-privileged niches" where they are protected from attack by the immune system. These "retreats" include joints, eyes and the CNS. These localizations contain extracellular fluids (synovial and cerebrospinal) that do not circulate in the lymphatic lymph flow. Furthermore, borrelia preferentially adhere to various cells. Borrelia adhere preferentially to various cells, such as thrombocytes, lymphocytes, fibroblasts, epithelia and endothelia. The BmpA protein (Borrelia membran protein A) appears to play an important role in the adherence capacity of Borrelia (Verma et al., 2009). Another mechanism of immune evasion is the ability of B. burgdorferi to bind the host's own complement regulators (Reconectin/FHL-1 and Factor H) to the cell surface.

Immune response: IgM antibodies are detectable in humans about 2-4 weeks after the initial infection. They reach their maximum about 6-8 weeks after infection. They then gradually fall again. Antibodies of the IgG class (IgG) appear after 6-8 weeks. IgM antibodies are often detectable over a longer period of time, which makes it difficult to draw conclusions about the approximate time of infection. The IgG concentration, on the other hand, can rise slowly over months/years as the disease progresses until it reaches a plateau and then remains relatively constant.

Note: It is not possible to distinguish between a clinically manifest and a survived Borrelia infection based on positive IgG detection. However, the detection of IgM-Ak alone largely rules out chronic Lyme borreliosis (Fingerle 2008).

Nomenclature of antigens: The name of the antigens is made up of the letter "p" (for protein) and their corresponding molecular weight in kDa (e.g. p39). Some antigens have their own names due to their function or localization, e.g. BmpA = Borrelia membrane protein A; OsP = "outer surface proteins". Osp are surface antigens of B. burgdorferi that form a fraction of heterogeneous lipoproteins, which are classified according to their molecular weight into the groups OspA to OspG; see below).

Serological screening: The patterns of anti-Borrelia antibodies in infected people show a high degree of variability, which makes their assessment difficult. Due to this complex immune response, it is necessary to use a panel of antigens for screening. This panel should contain proteins such as Osp C, BmpA = Borrelia membrane antigen = p39 antigen), p41 antigen and p100 antigen. A number of immunodominant Borrelia proteins could be assigned to the clinical stages of the disease.

Cross-reactivities/specificities: A number of conserved Borrelia proteins, such as heat shock proteins and parts of flagellin, have epitopes that are also found on other bacteria. This cross-reactivity naturally leads to false-positive results. Especially within the human pathogenic genospecies (B. burgdorferi s. s., B. afzelii, B. garinii and B. valaisiana) a pronounced heterogeneity was detected for OspA and OspC. For OspA, 7 different serotypes were defined. OspA serotype 1 corresponds to the species B. burgdorferi s.s., OspA serotype 2 to the species B. afzelii, and OspA serotypes 3-7 to the species B. garinii. A further serotype (serotype 8) has been defined for B. garinii. These differences are of great importance for diagnostics, as the different OspA serotypes are associated with different clinical manifestations in humans. Lyme arthritis, for example, occurs primarily in infections with B. burgdorferi s. s. Like B. garinii, B. burgdorferi is a trigger of neuroborreliosis. Skin manifestations such as acrodermatitis atrophicans, on the other hand, are mainly caused by infections with B. afzelii.

Important antigens:

• OspC: 22 serotypes of OsPC (outer surface protein) have been identified to date. While OspA (this lipoprotein serves the adhesion of the borrelia). OSPA is expressed in the gut of fasting ticks, the lipoprotein OspC is produced during the blood meal (Pal et al. 2004). The concentration of OspC increases when the spirochetes leave the tick midgut and invade the tick's salivary glands.
• OspC apparently plays an important role in the transmission process from the vector to the host (Pal et al. 2004). Like OsPA and OspB, the antigen is considered a very early marker for a Borrelia infection.
• VlsE: VlsE (variable major protein) is another surface lipoprotein that is important for the infection process, whose synthesis is also regulated by the sucking act and whose activation takes place during transmission to the host, is the plasmid-encoded VlsE. It can appear both in the early phase and the late phase of infection. VlsE appears to contribute significantly to the persistence of B. burgdorferi s.l. in the infected mammalian organism due to combinatorial antigen variation as an "immune escape" mechanism.
• BmpA (Borrelia membrane protein = p39): In addition to the proteins, OspA, OspC and p83/p100, the 39 kD antigen (BmpA) is also diagnostically relevant (Simpson WJ et al. 1990). This membrane-associated and immunodominant protein may be of great importance in the development of Lyme arthritis (Pal et al. 2008).
• FlaB Flagellin (P41): The flagellin-associated p41 antigen induces a very early immune response that occurs no later than five weeks after infection. P41 plays an important role in the diagnosis of early infection.
• Osp17 (syn. lipoprotein p17, decorin binding protein A, DbpA, p18): Osp17 is another immunodominant Borrelia antigen. The antigen, a lipoprotein, is localized on the surface of the outer membrane of the borrelia and is therefore also referred to as Osp17. Osp17 usually appears at a late stage and is considered a marker for a chronic course of infection (Jauris-Heipke et al. 1999).
• P58: P58 is another "outer surface protein" that is considered a late marker of infection. The protein is found in the three human pathogenic genospecies B. burgdorferi s.s., B. afzelii and B. garinii (Hauser et al. 1997).
• p83/p100: p83/p100, on the other hand, has structural properties that are similar to those of eukaryotic cells, resulting in an imitation effect that also contributes to immune evasion (Rössler et al., 1995). It is a late-emerging marker of infection.

Borrelia burgdorferi antigens (molecular weight in kDa in brackets) and their diagnostic relevance in the course of Lyme borreliosis (varies according to Müllegger B 2018).

Early stage of infection:

• FlaB Flagellin (P41)
• OspE (19,3) early/late
• OspC (22-24) very early
• VlsE p66 (66): Fusion protein, represents the immunodominant VlsE epitopes of different genospecies early/late
• p39 (39 ) BmpA
• p41 (41) Flagellin

Late stage of infection:

• Osp 17 (marker for chronic course of infection)
• p30 (30)
• OspA (31-33) B. afzelii surface protein
• OspB (34-36)
• p43 (43 )
• p45 (45)
• p58 (58)
• p83/100

After deposition in the skin, B. burgdorferi usually multiplies locally before spreading through the tissues and into the blood or lymphatic system, facilitating migration to distant sites. Motility (generated by the flagella) and adherence to host molecules (mediated by the surface lipoproteins) are critical for B. burgdorferi to move through the host's blood and tissues and evade immune responses. Mutant bacteria that exhibit defects in motility or chemotaxis are unable to spread and are rapidly cleared from the inoculation site.

Many of the mammalian phase-specific surface lipoproteins can interact directly with various host macromolecules, including plasminogen, complement-regulating proteins and extracellular matrix components such as fibronectin, collagen, laminin and glycosaminoglycans. It is assumed that these interactions have different functions, e.g. plasminogen for the proteolysis of tissues, extracellular matrix molecules for adhesion and complement-regulating proteins for immune defense. However, there is a high degree of functional redundancy between these B. burgdorferi lipoproteins. One exception is the fibronectin-binding protein BBK32, where unique and sequential roles are assigned to the fibronectin-binding and GAG-binding domains in the interaction of B. burgdorferi with the host vasculature. BBK32-fibronectin binding initiates the "tethering" of circulating B. burgdorferi to the vascular surface, and BBK32-GAG binding contributes to a more stable vascular interaction, after which B. burgdorferi migrates through the endothelium and spreads throughout the tissue.

B. burgdorferi utilizes multiple strategies to evade the host's innate and adaptive immune system.

Several lipoproteins of B. burgdorferi, known as complement regulator-acquiring surface proteins (CRASPs), can bind to host factor H, factor H-like protein and factor H-related proteins, which prevents complement-mediated killing of the bacteria in vitro.

Once the infection is established, evasion of bactericidal antibodies is crucial for bacterial survival. To this end, B. burgdorferi again modifies the lipoproteins expressed on its outer surface by replacing OspC with VlsE. Due to the structural similarities between OspC and VlsE, these two proteins may have a similar physiological function. However, unlike OspC, VlsE undergoes extensive antigenic variation to evade the host immune response.

B. burgdorferi can evade host antibodies against one VlsE variant by expressing another on the surface. Mutants expressing non-variant VlsE are unable to re-infect animals previously infected with B. burgdorferi, whereas bacteria expressing variant VlsE can.

The test result of the immunoblot reported by the laboratory should include an indication of the bands detected. For the evaluation of the borrelia serology, the level of AK in the EIA, the number of specific bands, the type of bands (early/late bands) and existing previous findings should be taken into account. The assessment should be carried out with knowledge of the clinical symptoms, the duration of the disease and therapies already carried out.

Diagnosis of neuroborreliosis: In acute neuroborreliosis, in contrast to most other infectious diseases, antibodies can initially only be detected in the CSF. For this reason, even if negative results are suspected in serum, the CSF must always be tested as well. The determination of antibodies in the CSF is only meaningful if a serum from the same day of collection is tested at the same time. Absolute antibody concentrations in the CSF are usually meaningless, since (especially if the CSF barrier function is disturbed) considerable quantities of antibodies can pass from the serum into the CSF. Autochthonously formed antibodies can be detectable for years even after neuroborreliosis has healed. In the case of neuroborreliosis, further pathological findings are usually found in the cerebrospinal fluid: monocytic cerebrospinal fluid pleocytosis; disturbance of the blood-cerebrospinal fluid barrier function (increase of the albumin quotient).

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