Clinical trials are vital and ongoing, particularly during the current pandemic. However, the risk from variants remains a concern.
A number of clinical pathologists from Cerba Laboratory recently presented some approaches on how to manage SARS CoV 2 variants during the clinical trial process.
Dr. Benedicte Roquebert is based in the microbiology department of Cerba Laboratory and works on emerging diseases, having authored 33 international publications on related topics.
Dr. Stephanie Haim-Boukobza has extensive experience working as a virologist in hospital and university settings, as well as private labs in France. She has been with Cerba since 2017 and became the Head of Infectology in 2018.
Dr. Souad Mehlal is a clinical pathologist who joined Cerba in 2017 to specialize in immunology and immunopathology and is now Head of Biochemistry, Immunology and Pharma-Toxicology. She has also authored numerous scientific publications.
Cerba Research is the pharmaceutical development arm of Cerba Healthcare. Cerba Healthcare is headquartered in Paris, France. The company employs over 8,500 staff, including over 130 pathologists, and conducts 40 million tests annually.
From pre-clinical transition to the clinic and onto commercialization, Cerba Healthcare supports emerging and established biotechs through to large global pharma clients. The company is well known for its close partnerships, frequent contact with and availability to clients.
What is a variant? What are the different types of variants?
Like all RNA viruses, SARS CoV 2 can mutate during each replication. Most mutations go unnoticed, but a mutation can lead to a new strain emerging. This is called a variant. There are different kinds of variants – the variant of concern, the variant of interest and the variant under monitoring.
A variant of concern (VOC) is a variant that can increase transmissibility or result in a non-favorable impact on SARS CoV 2 epidemiology. It may also increase clinical severity, adversely affect SARS CoV 2 control or prevention measures epidemic or it can impact a diagnosis test.
A patient infected with a variant of concern must isolate for longer, or it may be necessary to employ specific measures to manage the case.
A variant of interest (also known as a variant under investigation) typically features a mutation associated with phenotypical differences but not an increase in transmissibility. This kind of mutation can be responsible for transmission, multiple cases or clusters.
A variant under monitoring has no implications relating to virological, epidemiological or clinical changes, but it is important to monitor this kind of variant in order to follow its evolution and better understand the virus.
Besides variants, are there other ways that a virus can be classified?
Yes. Molecular classifications can be used to assign clades, lineage or mutations. The NEXSTRAIN software assigns clades with the number of the year (e.g., 19, 20), the arrival order (e.g., A, B, C, D) and the phylogenetic assignment of named global outbreak lineage.
For example, for the UK variant, the NEXSTRAIN and GISAID would be 20I/501Y.v1 of PANGOLIN or the B.1.1.7 – it is the same virus.
On May 6th, Public Health England (PHE) classified B.1.617.2 as a variant of concern because of the situation in both India and the UK. It was still considered a variant under investigation in France at that time.
Could you tell our readers more about key mutations and how these differ between variants?
The D614G mutation appeared in March 2020. This mutation was not present in Wuhan at the beginning, but it is now present in all the strains that we see in our patients. While this mutation can increase viral load in patients, there has been no clinical impact seen in the presence of this mutation.
The UK variant was discovered in Kent in September 2020. There were discrepancies in the detection of S target with the TaqPath Thermo Fisher assay, whereby a signal recognized a mutation. The Thermo Fisher assay detected a deletion (69/70) by recognizing an absent position that was present in this UK variant.
In the UK variant (B.1.1.7), there is a specific mutation that is very important – N501Y. This strain has been described as more contagious, with higher viral loads. Our team authored a publication outlining how this strain can lead to a more severe presentation and an increased risk of hospitalization and fatality.
The South African variant (B.1.351) was also discovered in September 2020. Mutations observed in this variant include N501Y like the UK variant, as well as 417N, which can impact binding to ACE2 receptors. The 484K mutation found in this variant has a negative impact on vaccine efficacy.
This strain can also increase transmissibility and present a more elevated viral load, but less than that of the UK variant. Clinical severity is increased, as well as the risk of hospitalization and fatality.
The presence of these mutations creates an environment that introduces probable resistance to immunization – both natural immunization and via vaccination.
The Brazilian (P1) variant was discovered in October 2020. Mutations observed in this variant are 501Y and 417T, which can impact binding to ACE2. The 484K mutation is also present, which can have an impact on spike shape.
Contagiousness, resistance to immunization, transmissibility and lethality could be increased, but studies are ongoing in this area.
There are many other variants, including the B.1.525 variant (which is seeing increased prevalence in France) and other lineages like the Californian lineage.
One variant currently causing concern is the B.1.617 variant from the Indian lineage. There are three different variants present in India, and this Indian lineage presents two specific mutations – 452R and/or the E484Q.
One of these variants – B.1.617.2 – is classified as a VOC by PHE due to its high levels of transmissibility. The majority of the strains in India are of this variant.
What impact do these variants have on epidemiology?
Initially, the 19A and 19B strains of SARS CoV 2 originated in Wuhan, with other strains evolving. Each time the majority strain changes, there is a wave of infections and vice versa. The question to ask is whether the strain provokes a wave or if the wave induces a new strain.
This question is difficult to answer because there is a delicate balance to consider.
On one side, we have factors such as barrier measures, confinement, valid testing and efficient contact tracing; and on the other, we have factors such as decreases in barrier measures, de-confinement, increasing strain contagiousness, testing or/and non-valid contact tracing or saturation of healthcare systems.
In summer 2020, some countries saw a decrease in barrier measures because of the holidays, and subsequently an increase in cases in August and September which caused the healthcare system to be completely saturated.
The more recent wave in December 2020 and January 2021 was different, however. There was no decrease in barrier measures, but the UK variant (B.1.1.7) appeared and was so contagious that a new wave occurred because of this.
The worldwide situation sees the USA, South America, Europe and India, as the regions most impacted. Meanwhile, countries like Israel, Australia and Japan are managing SARS CoV 2 due to high rates of vaccination.
What impact do variants have on test performance?
Mutation in spike genes can lead to problems in terms of signal detection. The Thermo Fisher TaqPath test mentioned earlier could potentially be affected by mutations because one of the signals was generated after amplification of a region at the same position as the 69/70 deletion.
Since this deletion was present in the UK variant, the signal could not be amplified.
These issues can lead to false negative results, particularly where a test cannot detect the presence of the currently circulating strains.
If a pathologist or virologist notices discrepancies when testing, it is important that they notify the FDA or an equivalent organization that there is a problem with a PCR signal. Three techniques typically present this problem: TaqPath, Accula and Linea assay, but it can occur with any test.
Due to this, it is essential to use a technique that amplifies multiple targets. If a test can only amplify one target, there is a possibility of a false negative result if the test encounters a variant it does not recognize.
How can variant screening assays help address this issue?
A variant screening assay is an assay that can confirm the presence of a specific variant, as well as providing vital information confirming the presence of a variant that presents less efficacy to a vaccine, for example.
Some mutations can also be screened via PCR signal – a technique that is very easy to implement and is similar to a multiplex assay. It does, however, offer less sensitivity than a screening assay, so this must be taken into account when interpreting the results.
Another useful type of assay is based on TEM. This technique is able to detect a mutation or a wild type strain by comparing hybridization temperatures.
We have performed thousands of screening tests per day in our laboratory, enabling us to publish detailed data documenting regional variations across France, including following the increasingly prevalent UK variant from January to February.
We began working on a SARS CoV 2 sequencing solution at Cerba Laboratory in May 2020. Our objective was not clinical diagnosis because we already had efficient tools, such as multiplex RT-PCR, with a capacity of 10,000 tests daily.
Instead, we wanted to be able to offer a solution to pharmaceutical companies that are monitoring clinical trials of new antiviral treatments or vaccine candidates.
The effects of variants and mutations are almost exclusively in terms of spike protein, in the receptor-binding domain which interacts with the cell receptor and on the N-terminal domain. The receptor-binding domain and N-terminal domain were present in the S1 part of the spike protein that was involved in the interaction between virus and host immunity.
The spike chain represents 1/10th of the whole genome sequence. It is possible to sequence a single path, the 29,903 nucleotides, of the SARS CoV 2 genome via Next Generation Sequencing (NGS).
The Sanger assay targets a single strand with fragments up to 1,000 nucleotides, while the S gene is around 3,821 nucleotides. We can sequence just one path of the S gene, but it is not possible to use a Sanger assay if several strains are mixed.
With Next Generation Sequencing, we can amplify and sequence short fragments, but this involves the generation of millions of fragments that are individually sequenced. This approach is more expensive, more complex and more technically demanding.
Despite these challenges, it is important to have the whole genome sequenced in order to classify variants into clades and to look for mutations potentially impacting the RT-PCR.
What are the typical considerations involved in a Next Generation Sequencing assay?
The stages between library preparation and clonal amplification are essential, and two protocols are possible: the capture-based approach or the amplicon-based approach.
The amplicon-based approach is faster and easier to work with, but it is subject to genome variation and lack of primer hybridization if there are mutations at primer positions. This could lead to sequencing gaps and inconsistent detection of new SARS CoV 2 mutations.
The capture-based approach takes longer and is more expensive, but it is not subject to genome variation because it tolerates up to 10 to 20% of mistakes. In order to assign clades and lineage, it is important to ensure appropriate coverage depths to guarantee coverage of the entire genome.
European Centre for Disease Prevention and Control (ECDC) provides guidelines around cladding and lineaging a segment, confirmation of reinfection and/or direct transmission, quasi-species haplotypes or/and detection of unknown pathogens or highly divergent strains.
The ECDC guidelines require sequences of around 5 to 10% of positive RT-PCR, with thresholds below 28 to be representative of a particular outbreak.
What is the impact of the variants on vaccination?
Vaccines such as Pfizer, Moderna and Comirnaty are made up of mRNA encoded spike proteins and devoid of infectivity, leading to the synthesis of anti-S antibodies.
The subunit protein vaccines, the conventional vaccines and the viral vector vaccines such as Vaxzevria by AstraZeneca, Janssen or Sputnik V are composed of a non-replicating viral vector that contains gene-encoding spike protein, prompting the synthesis of anti-S antibodies.
A number of vaccines are still able to affect variants with the D614G mutation or the UK variant, preventing the disease and preventing infection.
However, the South African variant (B.1.351) or the Brazilian variant (P.1) see vaccines becoming less effective, for example, the AstraZeneca vaccine only demonstrates around 10% efficacy in preventing disease with these variants.
Recent studies presented in the New England Journal of Medicine and The Lancet on infection among vaccinated people highlighted the new SARS CoV 2 infections among vaccinated healthcare workers from December 16th, 2020 to February 9th, 2021.
These studies showed that after one dose of the vaccine, there were new infections as no protection was in place, but 15 days after the second dose, there were very few new infections, confirming that vaccination is beneficial in preventing new infections.
In order to prevent the VOCs from spreading further, we have to vaccinate as soon as possible with existing vaccines. We also have to increase viral genome surveillance in order to adapt the vaccines to match the VOCs in the target region. These can be given as a first shot or booster shot until we are able to develop a universal SARS CoV 2 vaccine.
Why is it important to monitor immune markers in patients to help to predict severe clinical forms of COVID-19?
In the case of controlled disease (the majority of cases), alveolar macrophages, cytotoxic T lymphocytes and specific antibodies work to neutralize and eliminate infected cells.
In rare cases of uncontrolled infection, systemic inflammation can be triggered, especially by very high amounts of proinflammatory cytokines like interleukin 6. This phenomenon is known as the cytokine storm.
This unhealthy immune response can be monitored in a laboratory by quantifying the main proinflammatory cytokines like interleukin 6, interleukin-1 beta, interferon gamma and TNF alpha. Many clinical trials have shown the efficacy of using anti-cytokine or cytokine receptor blocking therapies in severe COVID-19 cases.
Another important pathway is the interferon pathway, which plays a key role in SARS CoV 2 infection. There are around 17 types of type I interferon, including alpha, beta, etc. These play a vital role in antiviral and antitumoral immunity and are secreted by many cell types, but primarily by plasmacytoid dendritic cells.
These interferons bind to their specific interferon (A, AR 1 and 2) receptors inducing the phosphorylation of the receptor by specific kinases and then the recruitment of STAT proteins which can control distinct gene expression programs for antiviral responses, anti-inflammatory responses and regulation pathways.
Unfortunately, SARS CoV 2 and other coronaviruses are able to escape this pathway by producing proteins like NSPs which enable virus recognition, ORF3a that inhibits binding to the receptor and ORF6, which inhibits the nuclear translocation of STAT1 and then inhibits interferon effects.
Approximately 80% of COVID-19 patients develop an efficient immune response and are asymptomatic or develop mild disease. Around 40% develop severe respiratory symptoms with dyspnea, and in rare cases, 5% of patients are critical with acute respiratory distress syndrome leading to admission to the intensive care unit, often culminating in multi-organ dysfunction and death.
Many risk factors have been identified, for example, age, obesity and diabetes. However, there is also an important individual variability in predicting the clinical evolution of these patients; and identifying these specific predictive markers has become a major focus of the work at Cerba.
How is Cerba working to identify these predictive markers?
First, we focused on the interferon signature, measuring interferon transcripts by RT-PCR, and calculating an ISG score. Many papers have highlighted the defect of the interferon pathway prevalent in critical patients, meaning that the ISG score could, therefore, be an indicator for severe COVID-19.
Tests for interferon signature are currently under development at Cerba.
Another interesting pathway involves the neutralization of interferons by the presence of specific autoantibodies. This was outlined in a paper published by Paul Bastard, who works in Jean-Laurent Casanova's team at the Imagine Institute in Necker Hospital in Paris.
Paul Bastard reports that at least 101 of the 987 patients with life-threatening COVID-19 pneumonia had neutralizing IgG autoantibodies. Thirteen of these patients had anti-interferon omega, 36 had antibodies specific to the 13 types of interferon alpha, and 52 had both types of autoantibodies.
These autoantibodies were not found in 663 individuals with asymptomatic or mild SARS CoV 2 infection, and were only present in four of the 1,227 healthy individuals.
Patients with life-threatening COVID-19 exhibit high titers of autoantibodies against type 1 interferons. Patient with autoantibodies were aged 25 to 87 years old, and the researchers saw a large predominance of men in this study.
Paul Bastard and his colleagues have shown that the presence of autoantibodies against interferon alpha-2 and omega neutralizes the effect of this cytokine in vitro and that the virus is able to replicate despite the presence of interferon alpha-2 in the presence of these autoantibodies. They also have shown that all types of interferon can be implicated.
A recent paper from Paul Bastard also demonstrated that these autoantibodies might be involved in other viral infections. The paper showed that they may result in adverse reactions to some vaccines like yellow fever vaccine – a live viral vaccine which may be used with patients positive for these anti-interferon antibodies.
This result suggests that these autoantibodies can be quantified in order to predict severe disease related to SARS CoV 2 infection. They can also be used to predict adverse reactions with some live viral vaccines while potentially playing a role in some autoimmune diseases, but this has yet to be demonstrated.
Cerba recently signed a collaboration contract with the Imagine Institute to continue this work and to identify the prevalence of these autoantibodies in the French population.
We are working to understand the origin of these autoantibodies and to determine if there is a genetic background. We are also working on the tech transfer and hope to develop a suitable test for patients and clinical trials.
Can you tell our readers about the significance of the plasmatic calprotectin marker?
Calprotectin is a heterodimer formed of two proteins – S100A8 and S100A9 – that is produced by activated monocytes and neutrophils in circulation and in inflamed tissues.
Calprotectin’s role in the inflammatory process is well known, especially in inflammatory bowel disease, where it is used in the diagnosis and as a predictive marker. Its role in the pathogenesis, diagnosis and monitoring of rheumatic disease has also attracted a great deal of attention in recent years.
Calprotectin could be a candidate marker for the follow up of disease activity in many autoimmune diseases, where it can predict the response to treatment or disease relapse.
The predictive role of calprotectin in COVID-19 has been described in several papers, including one by Aymeric Silvin from the Gustav Roussy Institute in France, who described several abnormalities in the myeloid compartment with large amounts of calprotectin produced.
Monomyeloid cells exhibit immunosuppressive phenotypes in severe COVID-19 cases with a predominance of HLA-DR low classical monocytes and impairment of the nonclassical CD14 low and CD16 high nonclassical monocytes.
There is also profound B and T lymphopenia and an accumulation of immature CD10 low, CD101 and CXCR-4 neutrophils with an immunosuppressive profile in the blood and the lungs, which suggests an emergency myelopoiesis.
This phenomenon correlates with significantly high levels of plasmatic calprotectin – higher than interleukin 6 levels. This data suggests that looking at the special phenotype of monocytes and neutrophils, together with the calprotectin plasma level, could provide robust biomarkers for severe COVID-19.
We have also signed a co-operation contract with the Gustave Roussy Institute to pursue this work on plasmatic calprotectin, and we are currently validating a potential test at Cerba.
Do genetic markers have a role to play in response to SARS CoV 2 infection?
A range of genetic markers have already been well characterized by the Jean-Laurent Casanova team, who created the COVID Human Genetic Effort – an international consortium aiming to discover the human genetic and immunological basis of the various clinical forms of SARS CoV 2 infection.
Zhang and their collaborators used a candidate gene approach to identify patients with severe COVID-19 who had mutation loss of function at 13 human loci known to govern toll-like receptor (TLR) 3 and interferon regulatory factor (IRF) 7-dependent type 1 interferon immunity to the influenza virus pneumonia.
They also described 23 COVID-19 patients with loss of function variant in TLR3, interferon IRF7 and interferon A receptor 1 and 2 with autosomal recessive or dominant transmission.
The patients were between 17 and 77 years old, and the team demonstrated that human fibroblasts carrying this mutation are vulnerable to SARS CoV 2. They, therefore, concluded that inborn errors of TLR3, IRF7 and interferon receptor 1 and 2, dependent type 1 interferon immunity could underlie life-threatening COVID-19 pneumonia in patients with no prior severe infection.
There is clear evidence to highlight the importance of the type 1 interferon pathway in the occurrence of severe clinical forms of COVID-19. The type 1 interferon response can be managed with biological testing techniques currently under development at Cerba, including autoantibodies against type 1 interferons and interferon signatures.
It has also been demonstrated that in severe cases of COVID-19, the myeloid compartment is disturbed, resulting in an immunosuppressive phenotype with the production of high amount of alarmins, such as plasmatic prolactin. Tests for the quantification of plasmatic calprotectin are under development at Cerba and will be available very soon.
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