New study - SARS-CoV-2 immunity after infection or not? Hide Article

A study by Long et al., recently published in the renowned scientific journal "Nature" (https://doi.org/10.1038/s41591-020-0965-6), strongly emphasizes why it is necessary, during and after the corona pandemic, to use readily available and efficiently performed antibody tests such as enzyme-linked immunosorbent assays (ELISA).

In the study mentioned above, Chinese researchers around Long and Huang compared samples from the megacity Chongqing. They examined samples of 74 patients with and without symptoms. After an infection, the body generates an immune response to eliminate a pathogen. The immune system first produces IgM and IgA antibodies. Once the pathogen is recognized and fought, IgG antibodies are produced. IgG antibodies offer some protection against future SARS-CoV-2 infections and are detected in the blood as so-called antibody titers. These IgG antibodies form the memory of the immune system for many years. Vaccines work similarly by teaching the immune system to produce antibodies to protect against specific pathogens.

Quantitative antibody tests that target neutralizing antibodies are among the most important tools for clinicians to determine a titer that reflects the value of a protective immune response to infection. Thus, the titer required for an effective vaccine can easily be determined using our SRS-CoV-2 AESKULISAs.

Long et al. have investigated this fact by measuring IgM and IgG levels in patient samples using ELISA. Even though the sample of patients is small, the study shows no insignificant decrease in antibodies in the patient group with mild to mild symptoms. Only 62.2 percent of patients in the group without symptoms still had antibodies in their blood a few weeks after infection. In the group of symptomatic patients, 78.4 percent still had antibodies in their blood.

After approximately eight weeks, follow-up examinations showed that the antibody concentration in the blood of the symptom-free patients decreased by 81.1 percent. In the group of symptomatic patients, the decrease was only 62.2 percent. The group of Long and Huang also identified patients positive for SARS-CoV-2 by ELISA, who had previously been categorized as negative by RT-PCR. An examination of the samples for cytokines involved in the immune reaction, including G- and M-CSF, IL2, IL6, CCL2, IFN-γ, shows that patients with symptoms had a higher value of these proteins, which indicates a stronger immune response.

Even though the study mentioned above only involved a comparatively small sample, the results cast doubt on previous assumptions that strong symptoms represent a high risk of infection and that anyone who has survived an infection is immune to future infections.

At Imperial College London, Professor Altmann confirms the present study, stating that most infected people show only mild or no symptoms at all. The crucial question is whether they have sustained protective immunity. For him, it is an essential but also worrying point, that many patients in the study showed a significant drop in antibody concentration only 6-7 weeks after the disease's onset. Long et al. write that the decrease in IgG and neutralizing antibodies in early convalescence will influence the immune strategy and serological investigations.

That is precisely where AESKU sees the primary clinical utility of its quantitaive AEKULISA SARS-CoV-2 tests and offers six different kits for detection and monitoring of the immune status.

 

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Tracing the Virus - AESKULISA SARS-CoV-2 Antibody Tests

A secure future thanks to quantitative SARS-CoV-2 AESKULISA tests

Corona VirusIn the current situation, it is hardly necessary to argue about the urgent need for an effective and robust vaccine to treat COVID-19, because our society and humanity itself are in a state of emergency. However, not only a vaccine is urgently needed, but the success of vaccination must also be verified using quantitative tests. Quantitative tests react to neutralizing antibodies, i.e., antibodies responding to a pathogen and breaking it down, make it possible to determine the success of an immune response and a vaccination in everyday practice.

For the determination of an immune reaction, the examination of immunoglobulins, so-called antibodies, has been established for years. Since protein structures on the surface of a pathogen (bacterium, virus) are specific, i.e., unique for individual structures, the immune system forms the appropriate antibody; it is possible to examine different antibodies for confirmation and infection. In the case of SARS-CoV-2 viruses, the nucleocapsid protein (N) and the spike protein (S1) are suitable.

 

AESKU offers six different immunoassays:

AESKULISA® SARS-CoV-2 NP IgM/ IgA / IgG*

AESKULISA® SARS-CoV-2 NP IgA*, IgG*, and IgM* tests are qualitative and quantitative immunoassays for the detection of human IgM, IgA, and IgG antibodies in serum or plasma directed against SARS-CoV-2 in highly conserved nucleocapsid protein (NP). The nucleocapsid protein is very reactive and stimulates a strong immune response. This potent immune response provides a very sensitive detection of antibodies, which allows a clear differentiation between positive and negative samples.

They are, therefore, ideal for IgM, IgG, and IgA detection for screening and diagnosis. Due to the high sensitivity and the strong conservation of the nucleocapsid protein in the coronavirus family, the risk of cross-reaction with antibodies against SARS-CoV-1 is increased. Since antibodies against the nucleocapsid protein have no neutralizing effect, they are not suitable for determining the immune status, the so-called antibody titer.

AESKULISA® SARS-CoV-2 NP IgA Antibodytest (Nucleocapsid Protein)
recommended for the detection of acute infections.
AESKULISA® SARS-CoV-2 NP IgGAntibodytest (Nucleocapsid Protein)
allows the confirmation of pathogen contact and the immune status's determination.
AESKULISA® SARS-CoV-2 NP IgMAntibodytest (Nucleocapsid Protein)
allows determining the first specific reaction of the immune system.

AESKU uses immunogenic nucleocapsid proteins of SARS-CoV-2 expressed in insect cells for the sensitive detection of IgM, IgA, and IgG antibodies.

AESKULISA®
Sensitivity
Specificity
SARS-CoV-2 NP IgA*
98.3 %
> 99 %
SARS-CoV-2 NP IgG*
95.2 %
> 99 %

Table 1: Sensitivity and specificity of AESKULISA® SARS-CoV-2 NP IgA
and IgG immunoassays were assessed by the analysis of 76 serum samples from healthy blood donors (2018) and 20 individuals with clinically confirmed COVID19 using the clinical findings as a reference.

AESKULISA® SARS-CoV-2 S1 IgM/ IgA / IgG

AESKULISA® SARS-CoV-2 S1 IgA, IgG, and IgM tests are qualitative and quantitative immunoassays for the detection of human IgM, IgA, and IgG antibodies in serum or plasma directed against SARS-CoV-2 by the highly specific spike protein (S1). The spike protein has the advantage over the nucleocapsid protein that is highly specific for the SARS-CoV-2 virus.

Resulting in a much lower risk of cross-reactions with antibodies against other members of the coronavirus family. IgG antibodies react against the receptor-binding domain (RBD) on the spike proteins. They are considered to be neutralizing and, therefore, suitable for monitoring the antibody titer of patients.

AESKULISA® SARS-CoV-2 S1 IgA Antibodytest (Spike Protein)
recommended for the detection of acute infections.
AESKULISA® SARS-CoV-2 S1 IgG Antibodytest (Spike Protein)
allows the confirmation of pathogen contact and the determination of the immune status. It can be used to detect and monitor the antibody titer of a patient.
AESKULISA® SARS-CoV-2 S1 IgM Antibodytest (Spike Protein)
allows determining the first specific reaction of the immune system.

* submitted for Emergency Use Authorization (EUA) in USA | IFU AESKULISA SARS-CoV-2 NP IgG USIFU AESKULISA SARS-CoV-2 NP IgM US

 

Asset 1 2Test Kit Components

  • Aluminium-sealed and coated MTP with breakable cavities
  • 4 Calibrators (A – D), Calibrator B = cut off Calibrator
  • Positive and Negative Control
  • Sample Dilution Buffer (5x conc.; for IgM detection incl. Rf Absorbent)
  • Wash Buffer (50x conc.)
  • Conjugate (anti-human IgG / IgA / IgM conjugated to POD)
  • Substrate (TMB) and „Stop Solution“
  • Quality Control Certificate and Instruction Manual

 

 

The AESKULISA SARS-CoV-2 immunoassays offer the end-user many unique advantages. These set them apart from the test kits of competitors:

 

  • Use of the immunogenic nucleocapsid protein for the detection of IgA and IgG antibodies directed against SARS-CoV-2
  • Standardized processing of IgA, IgG and IgM AESKULISA® with short incubation times (30 min, 30 min, 30 min) and consistent incubation temperatures
  • Optimized for incubation at room temperature
  • Combination of different AESKULISA® in one test run
  • Ready-to-use, colored, barcoded and interchangeable reagents for quality assured handling in routine laboratories
  • Cost efficiency by the use of breakable microtiter strips and a minimum number of calibrators and controls
  • Positive and negative controls according to modern quality management guidelines
  • Exact quantification of the pathogen-specific IgA, IgG and IgM antibody activity by use of the precise 4 parameter logistic (4 PL) function and 4-point calibration
  • Use of Cal B as cut off calibrator for qualitative data evaluation
  • Fast evaluation of measurement signals using standard software solutions
  • Excellent diagnostic efficiency with high sensitivity and specificity guaranteed by carefully selected antigens
  • High precision and linearity within wide measurement ranges guaranteed by a superior principle for antibody quantification
  • CE certified
  • Compatibility with conventional ELISA washer and reader systems
  • Complete automation on Triturus®, SQII, DSX and comparable instruments
  • Triturus®, SQII, DSX and comparable instruments can be optimally networked using HERA- the AESKU laboratory automation manager

Read more...

COVID 19
Severe acute respiratory syndrome (SARS)-CoV, as well as Middle Eastern respiratory syndrome (MERS) and SARS CoV 2 (SARS-CoV-2), belong to the family of Coronaviridae. All these three viruses are pathogenic and cause respiratory problems in humans. In December 2019, several unexpected cases of pneumonia were observed in China.

The government and health researchers in that country pursued strategies to control the virus's outbreak and programmed an etiological study. Finlay, the World Health Organization (WHO) announced that SARS-CoV-2 would cause coronavirus disease in 2019 (COVID-19).

AESKU Corona Virus open

The incubation period of COVID-19 is estimated to be 1 to 14 days. The lung is the main organ affected by COVID-19. However, in severe cases, other parts of the body, such as the central nervous system (CNS), kidney, liver, heart, stomach, and intestines may also be affected. As with many other respiratory diseases, the severity of the infection caused by SARS-CoV-2 can vary from patient to patient.

Recent evidence suggests that some SARS-CoV-2 infection cases have been associated with pneumonia and respiratory distress, while other patients diagnosed with SARS-CoV-2 have experienced respiratory failure, septic shock, or multi-organ failure. The mortality rate of infection is reported to be about 2%. The infection of other people during the incubation period and an acute symptom-free phase of the disease is possible.

Discovery

Discovery
Epidemiological studies of early SARS CoV-2 pneumonia cases have shown that in Wuhan, China, many cases have been in contact at the fish market. The WHO also reported that SARS-CoV-2 was found in samples collected from the fish market. However, it was not yet fully understood which specific animal species carry SARS-CoV-2. It is known that SARS-CoV and MERS-CoV originate from bats as the primary and natural reservoir and are transmitted from civet and camels to humans.

Taxonomy and structure

Taxonomy and structure
CoVs are 120-160nm large, enveloped single-stranded RNA viruses, sense-positive, of animal origin, belonging to the family Coronaviridae and the category Nidovirales. The CoVs are divided genotypically and serologically into four groups: α, β, γ, and δ. Approximately 30 CoV species have been identified in vertebrates, while in humans, CoVs from the groups α and β dominate. CoVs belong to the group of β CoVs, and SARS-CoV-2 is the third known zoonotic animal CoV after SARS and MERS viruses, both of which belong to the group of β CoVs.

In humans, there are at least seven CoVs (hCoVs): SARS-CoV-1 (SARS), MERS-CoV (MERS), SARS-CoV-2 (COVID-19, or simply COVID), and the endemic hCoV diseases NL63, HKU1, OC43 and 229E, whose corresponding viruses are designated by the suffix -CoV, as in MERS. For each of these hCoVs and CoVs in general, adsorption and fusion are influenced by spike glycoproteins.

Transmission

Transmission
The transmission from person to person is mainly aerogenic through the finest aerosols produced when breathing out and speaking (droplet infection). Direct contact with saliva, sputum, or nasopharyngeal secretions is also considered highly infectious. Since the virus has a proven survival time on surfaces of approximately 16 hours, infection via contaminated surfaces is also possible (smear infection).

Pathogenesis and replication

Pathogenesis and replication
The spikes for SARS-CoV-1, SARS-CoV-2, and NL63-CoV all bind to the angiotensin-converting enzyme 2 (ACE2), MERS-CoV to dipeptidyl peptidase 4 (DPP4), HKU1-CoV and OC43-CoV to 9-O-acetylated sialic acids and 229E-CoV to aminopeptidase N (APN). As determined by Clustal Omega, the spikes for SARS-CoV-1 and SARS-CoV-2 show a high homology identity (76%) but a relatively weak homology with NL63-CoV (30% and 31% respectively). The spikes for NL63-CoV and 229E-CoV have a surprisingly high homology identity (63%), with all other pairwise identities of these seven ranging from 30%-40%. MERS, SARS, and several endemic hCoV diseases appear to confer short- to medium-term immunity against reinfection. Endemic hCoV diseases are seasonal and favor the months of December and January, while MERS spreads mainly in June. The seasonality of COVID and reinfection immunity remain pressing open questions.

The CoV virus envelope has a diameter in the range of 120-160 nm, with each spike glycoprotein at the height of ∼15-20 nm above it. At the distal end of the viral envelope of the trimer spike are three heads, each with its receptor-binding domain, which is thought to be able to oscillate independently between an up (or upright/open) and a down (or lied/closed) conformation. Due to steric constraints, ACE2 binding can only occur in the up configuration. In hCoVs in general, there are three other domains of the spike, called lobes, which are near the heads when pointing up and adjacent when pointing down. These domains occur in the following pattern: The head in chain A (B or C, in that order counterclockwise when viewed from beyond the distal end of the virus envelope) is located proximal to the lobe in chain B (C and A). In the examples of MERS-CoV and SARS-CoV-1, which are attached to the viral envelope as described, the epitopes are also located on the heads.

The putative fusion peptide from SARS-CoV-1 lies centrally between the three heads and is completely blocked when the heads are folded down. After binding, the spike undergoes a dramatic reconformation; the details remain unknown. However, it eventually delivers the 6-helix bundle that is characteristic of a class I fusion protein. It is known that many of the hCoVs, including SARS-CoV-2, enter the cell through endocytosis. In several examples investigated, the spike is covered with an elaborate glycan shield. Both MERS-CoV and SARS-CoV-2, but not SARS-CoV-1, support a furin cleavage site that might promote fusion.

Symptomatology

Symptomatology
In addition to clinically completely inconspicuous infections, mild courses of disease with flu-like symptoms and dry cough have been documented to date. In some cases, severe courses of disease with severe pneumonia and cardiovascular symptoms have also been observed. Complications can occur, especially in high-risk patients with previous illnesses such as hypertension, diabetes mellitus, and obesity. In persons over 60 years of age, life-threatening conditions can occur, sometimes with a fatal outcome. In addition to severe damage to the lungs, up to and including lung failure, serious late effects in other organs such as the liver, kidneys, blood vessels, and the heart, as well as permanent damage to the central nervous system have been described.

Clinical Symptom
Frequency
Fever
87,9 %
Dry cough
67,7 %
Discomfort and fatigue
38,1 %
Increased saliva production
33,4 %
Smell loss
30 - 71 %
Shortness of breath
18,6 %
Muscle or joint pain
14,8 %
Sore throat
13,9 %
Headache
13,6 %
Chills
11,4 %
Nausea / vomiting
5,0 %
Common cold
4,8 %
Diarrhoea
3,7 %
Hemoptysis
0,9 %
Conjunctivitis
0,8 %

Therapy and prophylaxis

Therapy and prophylaxis
A specific therapy does not exist so far but is currently under development (Status 20.05.2020). Alternatively, anti-virals such as protease inhibitors and protease inhibitors of RNA are used. All currently applied therapies aim to alleviate the symptoms.

Immune response and antibody profiles

Immune response and antibody profiles
IgM, IgG, and IgA antibodies are produced during primary infections with pathogens, in this case, SARS-CoV-2. The organism can identify pathological structures of an intruder (viruses, bacteria) and form a tailor-made immune response, the so-called "key-lock principle." These antibodies are differentiated according to the place and time of their appearance in an immune response. IgM and IgA are the classes of antibodies that the body forms first, i.e., antibodies of the acute phase. IgM and IgA are no longer detectable within a few weeks to months after infection.

Antibodies of the G class, i.e., IgG, form the "memory" of the immune system and are usually detectable throughout the patient's life. They are only formed when the acute phase of infection subsides and persist longer in the blood. In case of re-infection, IgA and IgG antibodies are formed very quickly, while IgM antibodies are often not detectable

Virus load