An international team of scientists from China, Saudi Arabia, and the United States in July 2020 published the results of the study:

  • the kinetics of virus reproduction in patients with COVID-19;
  • distribution of viral load across tissues;
  • neutralizing antibody response;
  • cross-reactivity with other coronaviruses.

The study involved two groups of patients with a positive PCR test for COVID-19.

The first group – 12 people, patients with severe COVID-19. These people needed artificial ventilation and were in intensive care units. All were diagnosed with severe pneumonia.

The second group – 11 people, patients with a mild form of COVID-19. These people did not need intensive care. Their clinical picture included fever, cough, malaise, headache, mild pneumonia.

Most of the patients were men (83%) older than 50 years, and the average age was 56 years (24-82 years).

Scientists carried out sampling and laboratory analysis of biological material every 3 to 4 days. To study the lesion’s temporal profile of different tissues, the researchers studied: 84 smears from the nose, 59 smears from the throat, 36 sputum samples, 90 fecal samples, 79 urine samples, 113 blood plasma samples, 1 sample of gastric juice. A total of 461 samples were studied.

Dynamics of viral load in different tissues and duration of coronavirus RNA isolation

In most patients with severe disease, virus isolation occurred within 30 to 40 days from the study’s start. In most patients with mild COVID-19, the virus stopped being detected after 15 days.

The peak viral load was significantly different in both groups. In respiratory samples (smears from the nose and throat, sputum), the viral load was higher in the group of severe patients. Also, in this group, viral RNA with feces was released longer. Periodically, urine and plasma were also COVID-19-positive:

Figures content:

  • X-axis: days from the beginning of the study.
  • Y-axis: depersonalized patient data. PT1-PT12: severe patients from the intensive care unit. PT3-PT23: patients with mild disease.
  • Horizontal dotted line: a border between different groups of patients.
  • Colored rectangles of varying degrees of intensity: a heat map of the viral load’s Ct values. The brighter it is, the higher the viral load. Correspondence of the heat scale to the one-dimensional digital graph at the bottom. Ct < 37 means that coronavirus RNA was detected in the sample.
  • Graph A: Analysis of nasal smears.
  • Graph B: Analysis of pharyngeal swabs.
  • Graph C: Sputum analysis.
  • Graph D: Fecal analysis.
  • Schedule E: analysis of urine.
  • Graph F: Blood plasma analysis.

The overall dynamics of IgM and IgG antibodies production in COVID-19

To establish the kinetics of the antibody response against the SARS-CoV-2 coronavirus that causes COVID-19, the scientists analyzed IgM and IgG antibodies’ responses to the N-proteins of the coronavirus RNA envelope. Blood plasma of patients was used for the study.

IgM antibodies in patients with severe COVID-19 increased within 1 to 2 weeks from the study’s start. After 4 weeks, the antibodies gradually decreased.

In patients with mild disease, IgM antibodies were significantly lower. The majority of patients in this group (8/11) did not produce a significant amount of such antibodies during the entire disease period. Scientists emphasize that the diagnosis of IgM for patients with mild disease is not sufficient.

IgG antibodies appeared after 10 to 15 days. Most patients had high levels of these antibodies, which persisted for at least a month and a half.

To ensure the test’s reliability, the researchers examined the plasma of a healthy donors’ control group: 48 samples taken in 2017-2018. No specific antibodies to SARS-CoV-2 were found in this group.

Figures content:

  • Y-axis: index of the number of antibodies. The principle of determining the index: the analyzed serum is placed in a laboratory tablet’s wells covered with a purified COVID-19 antigen. If there are specific antibodies, they bind to the antigen. Unbound antibodies are washed away, and what remains is incubated at 37°C. After incubation, the color (optical density – OD) of antibodies associated with the antigen changes. The color change is determined photometrically at a wavelength of 450 nm. Interpretation of the result: the brighter the color (higher the index value), the greater the number of specific antibodies in the sample.
  • The horizontal dotted line on the chart: a detection threshold of the antibodies. The graph below the dotted line — no antibodies detected. The graph above the dotted line — antibodies detected, the higher the value, the more antibodies.
  • Graphs A: the presence of IgM antibodies to SARS-CoV-2. A first schedule is a group with a severe form of the disease. A second graph is a group with a mild form of the disease. The third is the control group: HD-healthy donors. PC — positive control, NC-negative control.
  • Graphs B: the presence of IgG antibodies to SARS-CoV-2.

IgM and IgG antibody production dynamics in different tissues

To investigate antibodies’ presence to SARS-CoV-2 in the tissues associated with the virus’s release, the scientists analyzed different types of patients’ biological material. Samples of sputum, bronchoalveolar fluid, pleural effusion, urine, and feces were examined. As a result, responses of IgM and IgG antibodies to the SARS-CoV-2 nucleocapsid protein were revealed.

In seriously ill patients, virus-specific IgMs were found in urine (3/10) and sputum (4/10). IgG antibodies were present more frequently in this group of patients: in urine (7/10) and sputum (7/10). In one of the severe cases, IgM and IgG antibodies were found in the bronchoalveolar fluid and pleural effusion. Based on the data obtained, scientists conclude that a severe coronavirus infection can damage various tissues, such as the respiratory tract and kidneys.

In the group of patients with mild disease, antibodies to SARS-CoV-2 were not found in the studied samples.

In both groups, there were no antibodies in the fecal samples.

Scientists suggest that the appearance of IgG to SARS-CoV-2 in urine and sputum may be a potential marker for determining the severity of the disease.

Figures content:

  • Graphs A: the presence of antibodies in urine samples. The first graph is IgM antibodies in patients with severe COVID-19. The second graph is IgM antibodies in patients with mild disease. The third and fourth graphs show the presence of IgG antibodies.
  • Graphs B: the presence of antibodies in sputum samples. The first graph is IgM antibodies in patients with severe COVID-19. The second graph is IgM antibodies in patients with mild disease. The third and fourth graphs show the presence of IgG antibodies.
  • Graphics C: the presence of antibodies in the stool samples. The first graph is IgM antibodies in patients with severe COVID-19. The second graph is IgM antibodies in patients with mild disease. The third and fourth graphs show the presence of IgG antibodies.
  • Graphs D: the presence of antibodies in samples of bronchoalveolar fluid and pleural effusion. The first graph is IgM antibodies. The second graph is IgG antibodies. HD-healthy donors. PC — positive control, NC-negative control.

Dynamics of antigenicity of SARS-CoV-2 proteins

The antigenicity of a viral protein reflects the strength of the immune system’s response to it. The higher the antigen, the more antibodies are produced against a particular protein.

Scientists compared the antigenicity of various SARS-CoV-2 proteins:

  • S-spike protein of coronavirus;
  • S1 is a fragment of the S spike protein responsible for binding to the cell;
  • RBD is an S1 fragment that directly binds to the ACE2 cell receptor.
  • S2-fragment of the spike protein S, responsible for penetration into the cell;
  • N – protein envelope of the viral genome.

The researchers used the same method for determining the antibody count index as in the experiment to calculate the overall dynamics of IgM and IgG antibody production. Blood plasma of patients was used for the analysis. The researchers covered the wells of the laboratory tablet with antigenic proteins. They then used the patients ‘ plasma to determine the antibody count index.

As a result, it was found that all proteins: S, S1, S2, RBD, N – were recognized by antibodies in the blood plasma. The peak of recognition was reached 3 to 4 weeks after the onset of the disease.

Recognition of protein S and S2 reached a maximum after 7 to 14 days. Recognition of S1, RBD, and N proteins peaked 3 weeks after the onset of the disease.

The researchers noted no apparent differences in IgG response against viral proteins between seriously ill and mild patients.

Additionally, the relationship between IgG antibodies’ levels against different viral proteins was calculated: S, S1, S2, RBD, N. Scientists found that most IgG responses showed a strong or moderate correlation. There was no correlation between IgG vs. S2 and IgG vs. S1 and N.

Figures content:

  • Graph A: response of IgG antibodies against S protein. The horizontal dotted line on the chart: the detection threshold of the antibodies.
  • Graph B response of IgG antibodies against S1 protein.
  • Graph C: IgG antibody response against RBD protein.
  • Graph D: IgG antibody response against S2 protein.
  • Graph E: Response of IgG antibodies against N protein.
  • Graph F: Heat map of the relationship between IgG antibody levels against different viral proteins. The brighter the color — the higher the relationship.

Cross-reactive responses of antibodies to different coronaviruses

To date, 7 known coronaviruses infect people and cause respiratory diseases: SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-OC34, HCoV-229E, HCoV-HKU1, HCoV-NL63.

Scientists have estimated how much antibodies after COVID-19 will be able to protect against other coronaviruses. For comparison, the spike (S) and nucleocapsid (N) proteins of the other six coronaviruses were selected. Healthy donor plasma (HD) was used as a control group.

As a result, it was found that the plasma of patients from both test groups recognizes viral proteins of coronaviruses SARS-CoV, HCoV-OC34, HCoV-229E, HCoV-HKU1, HCoV-NL63. In only one case, there was no apparent cross-reactivity between antibodies specific to SARS-CoV-2, which causes COVID-19, and MERS-CoV, which causes Middle East respiratory Syndrome:

Figures content:

  • Red dots — plasma of patients with severe COVID-19.
  • Blue dots — plasma of patients with mild COVID-19.
  • Black dots — plasma of the control group of healthy donors.
  • Green dots — control group plasma for positive control.

The kinetics of the neutralizing activity of antibodies to SARS-CoV-2

The researchers evaluated the dynamics of neutralizing antibodies to SARS-CoV-2 in patients with severe and mild COVID-19. Blood plasma of patients was used for the analysis.

It was found that 73.9% of patients produced stable neutralizing antibodies 3 weeks after the onset of the disease. Higher titers of neutralizing antibodies were found in the group of seriously ill patients. Plasma samples collected from patients 3 weeks after the onset of the disease still showed neutralizing antibodies.

A plasma of recovering patients with MERS-CoV and SARS-CoV did not contain neutralizing antibodies to SARS-CoV-2. Therefore, scientists conclude that patients with MERS and SARS may still be vulnerable to COVID-19.

There was also no apparent correlation between viral load and the amount of neutralizing antibodies.

Figures content:

  • Graph A: dependence of the number of neutralizing antibodies on the disease’s duration for a group of severe patients.
  • Graph B: dependence of the number of neutralizing antibodies on the disease’s duration for a group of patients with mild disease.
  • Y-axis in graphs A and B: The test’s value evaluates the number of neutralizing antibodies by reducing the lesion by 50% (FRNT50).
  • Graph C: Comparison of cross-neutralizing antibodies against the SARS-CoV-2 pseudotype.
  • Graph D: Comparison of cross-neutralizing antibodies against authentic SARS-CoV-2.
  • Red dots on graphs C and D: antibodies isolated from the plasma of patients with severe COVID-19.
  • Blue dots on graphs C and D: antibodies isolated from the plasma of patients with mild COVID-19.
  • Green dots in graphs C and D: antibodies isolated from the plasma of patients infected with SARS-CoV.
  • Purple dots in graphs C and D: antibodies isolated from the plasma of patients infected with MERS-CoV.
  • Black dots in graphs C and D: data for the control group of healthy donors.
  • Graph E: Evaluation of the relationship between the neutralizing titer and S-specific IgG levels.
  • Graph F: Evaluation of the relationship between the neutralizing titer and N-specific IgG levels.
  • Graph G: The viral load Ct of respiratory samples on the number of detected neutralizing IgG antibodies to SARS-CoV-2.

Source

Kinetics of viral load and antibody response in relation to COVID-19 severity

 

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