Development Of Antibody Response To Hsv And Vzv Biology Essay

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Herpes simplex (HSV) and varicella-zoster (VZV) viruses are common human pathogens with a worldwide distribution that has been associated with a number of clinical manifestations ranging from a mild illness to potentially fatal viral encephalitis (1). The diagnosis of viral encephalitis requires concerted, multidisciplinary approaches including clinical evaluation, cerebrospinal fluid (CSF) examination, detection of viral antigens or nucleic acids, imaging studies and examination for intrathecal production of specific antibodies in the CSF. During acute herpes simplex encephalitis (HSE), virus DNA can be detected by polymerase chain reaction (PCR) in CSF and often disappears after one-to-two weeks of treatment (1). PCR is a sensitive test, but is not infallible; false-negative test results have been observed due to limited virus replication and presence of PCR inhibitors in CSF (2). Although, acute VZV encephalitis is very rare, acute disseminated encephalomyelitis (ADEM) occurs in one per 1000-5000 cases of varicella (3). PCR is utilized in acute VZV encephalitis, but it is not used in diagnosis of the much more common neurological diseases associated with varicella zoster such as Acute Disseminated Encephalomyelitis (ADEM). ADEM is an example of an autoimmune disease targeted by a virus infection. Therefore, the virus DNA can not be detected in this condition. However, an intrathecal humoral immune response occurs following VZV infection of the brain and the appearance of VZV antibodies within the CNS compartment allows diagnosis of both acute varicella encephalitis and ADEM.

Two techniques are commonly used to detect antigen specific IgG in CSF: measurement of antibody in serum and CSF with calculation of specific-antibody indexes, and antigen-specific immunoblotting of oligoclonal IgG. Whilst these assays are useful in confirming antigen-specific antibody within CSF, they are labour intensive, require replication with multiple different antigens, and are time consuming. Since encephalitis is a life-threatening disease, a simple and sensitive assay with a rapid turnaround time would be helpful to screen for the presence of antigen-specific antibodies in the CSF.

In our current study as an effort to develop an assay with increased sensitivity and specifity for the detection of HSV and VZV antibody in human sera and CSF samples, we investigate the visibility of using a recombinant HSV-1 gD, VZV gE proteins and an HSV-2 specific truncated chain oligopeptide peptide 55 to develop a multiplex florescent microbeads immunoassay for the stimulus detection and quantitative measurements of these viruses antibody in human sera and CSF samples.

Methods:

Recombinant proteins (HSV-1 gD and VZV gE) expressed in insect cells using a novel plasmid expression system (InsectDirect, Novagen, Merck, USA) were used in the developed assay. The full open reding frame of each DNA region encodes HSV-1 gD and VZV gE were amplified by PCR and cloned into pIEx/Bac-3 3C/LIC plasmid expression vector using the ligation-independent cloning (LIC) strategy. Insect cells (Sf9) were transfected with the recombinant plasmid and 48h post infection cells were harvested and the recombinant proteins were solubilised, purified and characterised using immunoblot and ELISA. For the differentiation between HSV-1 and HSV-2 an immunodominant epitope of glycoprotein G2 presented in a branched chain format (peptide 55) was used (2).

An in-house ELISA for each recombinant protein were developed and optimised first by chalkboard titration and based on the optimisation results a microbead florescent immunoassay was developed. Using a standard modified two step carbidiimide reaction, each antigen (HSV-1 gD, VZV gE and peptide 55) were coupled directly to the different SeroMAP carboxylated microspheres. A monoplex assay for each antigen was developed and optimised individually and then the three assays were mixed in a triplex assay. Assay reproducibility was determined by measuring intra and inter assay variation using 10 samples tested in duplicates within the same plate in the same day or in two different days. The assay was then evaluated for the detection and measuring of antibody response to these viruses by testing a well characterised serum panel consisting of 218 human serum samples. Assay Sensitivity, specificity, positive and negative productive value was calculated using Western blot assays as a reference test for HSV-1 gD, Peptide 55 and Liaison VZV IgG assay for VZV gE.

Results

A microbead fluorescence immunoassay for stimulus detection and measurement of HSV-1, HSV-2 and VZV antibody levels in human serum was developed. Results showed that of the 218 samples tested 184 were found to be HSV IgG antibody positive and 34 were negative for HSV antibody. A total of 47 of the 184 HSV positive samples were positive for antibody against peptide 55, indicating HSV-2 IgG positivity (Table 1). For VZV, 202 out of 218 samples were determined VZV IgG antibody positive (Table 2). Comparison of the results obtained with this assay with different ELISAs previously used to characterise the panel showed sensitivity of 100%, 100% and 97.9 % for HSV gD, VZV gE and peptide 55 respectively. Intra and inter-assay CVs were generally less than 10%. Assay results were also plotted to generate a scattergram, regression analysis (r2) results are shown in Figures 1-3.

TABLE 1. Multiplexed Fluorescence Microbead Immunoassay Results in Comparison to Western Blot Assay.

Western Blot Assay

Results of different Analyte Obtained from the Multiplex Fluorescence Assay

HSV gD Assay

Peptide 55

Positive

Negative

Equivocal

Positive

Negative

Equivocal

Positive

184

0

0

47

1

0

Negative

0

34

0

0

34

0

Equivocal

0

0

0

0

0

0

Total

218

Sensitivity: 100% and 97.9% respectively, for HSV gD and Peptide 55.

Specificity: 100% and 100% respectively, for HSV gD and Peptide 55.

Positive Predictive Value: 100% and 100% respectively, for HSV gD and Peptide 55.

Negative Predictive Value: 100% and 97.1% respectively, for HSV gD and Peptide 55.

Table 2 Multiplexed Fluorescence Microbead Immunoassay Results in Comparison to DiaSorin VZV IgG Assay.

DiaSorin VZV IgG Assay

HSV-1 gD Fluorescence Microbead Immunoassay

VZV gE Assay

Positive

Negative

Equivocal

Positive

202

0

0

Negative

0

16

0

Equivocal

0

0

0

Total

218

Sensitivity: 100% Specificity: 100%

Positive Predictive Value: 100% Negative Predictive Value: 100%

Fig 1 The figure shows the results of regression analysis on measured serum HSV-2 antibody concentration between Peptide 55 ELISA (χ axes) and Peptide 55 florescence microbead immunoassay (у axes). The measurements were preformed on 218 sera samples; regression was 98.45 % (R2 regression coefficient).

Fig 2 The figure shows the results of regression analysis on measured serum VZV antibody concentration between VZV gE in-house ELISA (χ axes) and the VZVgE florescence microbead immunoassay (у axes). The measurements were preformed on 218 sera samples; regression was 96.27 % (R2 regression coefficient).

Fig 3 The figure shows the results of regression analysis on measured serum HSV antibody concentration between HSV-1 gD in-house ELISA (χ axes) and the HSV-1 gD florescence microbead immunoassay (у axes). The measurements were preformed on 218 sera samples; regression was 95.9 % (R2 regression coefficient).

Conclusion:

A sensitive and specific multiplexed fluorescence microbead immunoassay was developed and evaluated against commercially available ELISA assays. The developed assay showed improved sensitivity compared to existing assays and had the advantage of allowing the detection of an antibody response against these three herpes viruses simultaneously in a single reaction. The technology has the potential to allow simultaneous detection of in excess of 25 different antibodies within the same sample, leading to reduced sample requirements (2µl) and faster and more comprehensive serodiagnostic testing.

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