Using Genomic Sequencing to Contain COVID-19

If you haven't noticed already, the game has moved on, subtly but surely. Countries are moving on from lock downs, even as cases are at best stable or slowly declining (UK, USA, Italy, Spain), or even surging in some (Brazil, India, Russia). There is now a tacit acceptance that the price of continued and total lock down is too steep, and that nations may have to accept some new cases, in order that society and economy as a whole, can reopen.

It is not a question of if but when. Cases will surge in many places. The pandemic hasn't peaked in the last 3 nations, and is hardly under control in the first group. Some countries have rolled out a "track and trace" mobile app, based on using bluetooth signal to inform if the phone in question has been in the vicinity of one owned by a COVID sufferer. For this, you have to download a track and trace app. If your phone data shows you are at risk, a "track and tracer" (25000 strong in the UK) will contact you, and ask you to self-isolate for 14 days. If you develop the disease, you self isolate for 7 days.

However, there is another way- something that makes the track and trace much, much more effective. This is the power of genomic screening of the COVID19 strains. RNA viruses undergo many more mutations than DNA viruses. One reason for this is the spontaneous deamination of cytosine to uracil. When this happens in DNA virus, this is quickly detected by the virus, and the base is corrected back to cytosine, as uracil is not normally present in DNA. However, this cannot happen in RNA viruses, as uracil is not a "foreign base", and will therefore not be "corrected". C to U mutations will therefore accumulate.

In fact, RNA viruses have over 10 times the rate of mutations as DNA viruses. This does not increase or decrease the pathogenicity of the virus appreciably, but it does mean that within a community or a nation, there might be several "strains" (with differing genome sequences) of the virus in circulation. As this usually affects only a single nucleotide, rather than blocks, it is called "intra-host single nucleotide variation", or simply iSNV. And this presents an opportunity for those seeking to track the spread of the virus.

Consider this. With a limited number of cases, you have the power to sequence the genome of the virus in every documented case within a matter of hours. Each cluster of cases will have a "signature" viral genome, because of the fact that the index case will have a viral strain with its own unique mutations. Thus, once the pandemic is stable and reasonably contained, scientists have within their power to look at the viral genome of a new case, and from a database of existing patients, pinpoint exactly from whom the infection was acquired. Thus, self isolation, instead of being a non-selective and disruptive tool, can be applied selectively and in a limited fashion, to maximum effect. The rest of the population can get on with their lives.

Some examples of common viral mutations might make this easier to understand. During the Zaire Ebola epidemic, it became clear that within the incorporated viral genome in host DNA, a disproportionately high number of mutations were thymine to cytosine (T>C). (Thymine does not appear in RNA, so this is referring to the host DNA that has incorporated the complementary sequence of the viral RNA). It turns out that the preponderance of T>C in virus infected cells is due to the action of an interferon inducible enzyme called "Adenosine deaminase acting in RNA 1", or simply ADAR1. (Interferons, as you know, are produced by the host in response to viral infections).

ADAR1 deaminates adenosine to inosine. Inosine is not a natural nucleotide, and is read as guanosine by the cell. Since the complementary base of guanine is cytosine, the thymine bases complementary to adenine are "corrected" to cytosine by single nucleotide excision and repair. Hence T>C.

This is not a pipe dream. Scientists in NZ, Australia and UK have an extensive database of viral genomes circulating in their respective nations. While the database is virtually 100% complete in NZ and Aus, the UK has data on 20% of viral genomes in circulation, given the very large number of cases. But they are getting there.

This, IMO, presents the only realistic way of opening up the society while continuing to promptly identify and isolate cases and their contacts.

Are Camelids the Key to Beating COVID-19?

The normal antibody (immunoglobulin) has two light chains and two heavy chains. Each chain, be it light or heavy, has a variable and a constant fragment. The variable fragment binds to the putative antigen, while the constant fragment carries the Fc receptor that lets cells like NK cells and neutrophils bind to the antibody. The constant portion also binds and activates complement through the classical pathway.

in 1984, Raymond Hamers at the VUB university in Brussels, while analysing the blood of dromedary camels for antibody response to a Trypanosomal species (the dromedary camel is the Arabian camel, with a single hump, as opposed to the double humped Bactrian camel, found in the plains of Central Asia), found to his surprise that the camel antibodies did not look like the human counterparts at all. They lacked the light chain altogether, and contained only the heavy chain, comprised of the variable and heavy fragments. Quite appropriately, these antibodies were named "camelids".

The 1990s saw the establishment of phage display libraries, which allow the manufacture of virtually any antibody in bacteria, by inserting the relevant sequence in bacteriophages, which then infect the bacteria, and uses the bacterial enzymes to make the protein whose sequence has been inserted into the phage. This technique is responsible for producing most monoclonal antibodies these days, having moved on from the days when antibody producing B-cells were immortalised by fusing them with rat myeloma plasmablasts, a technique described as hybridoma.

It is now possible to produce through such phage display techniques, not just whole immunoglobulin molecules, but parts thereof, such as a the Fab fragment (commercially marketed as Certolizumab), a single chain variable fragment, the camelid (commercial application Caplacizumab, used to treat acquired thrombotic thromobocytopenic purpura), or an isolated variable heavy chain fragment, called VHH. Please see Diagram.

It is the VHH, or the variable heavy chain single domain antibody that now provides promise for the treatment of COVID-19. While vaccines can take years, and canonical (standard) monoclonal antibodies around 6 months to prepare, VHH can be prepared very quickly- within weeks, and therefore are ideally suited for dealing with a pandemic. Please see the linked paper in Cell below:

https://www.cell.com/cell-host-microbe/fulltext/S1931-3128(20)30250-X

VHH has several advantages over other techniques. It ways around 15 kDa, around a tenth of the full immunoglobulin molecule. It can reach antigenic epitopes that the full immunoglobulin molecule cannot reach, such as hidden epitopes, a fact that is relevant with COVID-19. And it does not have the foreign antigenicity that full camelids have, thus reducing the risk that they would be rendered ineffective by the human immune system.

Is Re-infection By COVID-19 Possible?

Once you have had COVID19, can you be reinfected with the same virus?

This article in JAMA may provide some reassurance, although it's understandably based on very little data.

https://jamanetwork.com/journals/jama/fullarticle/2766097

If you wanted a summary, it's halfway down the article, in this line here:

To date, no human reinfections with SARS-CoV-2 have been confirmed.

However, as with everything COVID, answers may not be forthcoming for a very long time. Hence, you look at the literature to see if there are predictors of long term immunity after infections, and a couple of facts begin to emerge.

In general viruses (excluding Influenza, which is prone to antigenic shift and drift) tend to cause long lasting immunity- the half life for antibody levels is between 50-200 years, from a 26 year long study of subjects following infections by 6 viruses- Vaccinia (the virus used for small pox vaccine), measles, mumps, rubella, Varicella zoster (chicken pox), and EBV. This may explain why there has never been a sustained second epidemic by a non-influenza virus, i.e. SARS, MERS, Marburg, Zika, Lassa Fever etc (don't misinterpret this as meaning that these viruses cannot infect naive subjects).

https://www.nejm.org/doi/full/10.1056/NEJMoa066092

The same does not apply to antibody responses to protein antigens, specifically for bacteria. For example, the half life for antibodies to Tetanus and Diphtheria is only 11 years-a fifth of that to the virus with shortest lived immunity.

Thirdly, women tend to have longer lasting protection than men.

https://genomemedicine.biomedcentral.com/articles/10.1186/s13073-018-0568-8

Fourth, if you have low antibody levels to start with, for example in a condition called Common Variable Immunodeficiency, a predominantly IgM response to the infective agent would suggest that the response will not be long lived. This may sound strange, but is intuitive, as IgM is the first antibody isotype produced for any sort of humoral (antibody mediated) response. It is then gradually followed by an IgG response. If IgG (particularly IgG1- there are 4 subclasses of IgG-1, 2, 3 and 4) levels do not increase appreciably, the response will not be long lived. Therefore in general, a high IgM level and a low IgG level- particularly that for the IgG1 component- is a worrisome feature.

https://www.jacionline.org/article/S0091-6749(18)30560-8/fulltext

And finally, some people use memory B cells as a surrogate marker for long lasting immunity. This is not a correct assumption. Long lasting humoral immunity can be memory B cell dependent and memory B cell independent, and each exists independently of the other.

If you wanted a straight yes or no answer to the question as to whether infection with COVID-19 is likely to lead to long lasting immunity- I would say, based on the available evidence, the answer is "Yes".

Is COVID-19 Transmitted Sexually?

Recently, concerns have been raised by reports that COVID-19 could be transmitted sexually, based on its presence in semen by RT-PCR. However, caution must be exercised in drawing any conclusions about sexual transmissibility, based on the above.

It's instructive that the original SARS virus uses the same ACE2 receptor as COVID-19, and in 20 years since it first affected humans, not a single case of sexual transmission (based on epidemiologic or molecular studies) has been described for SARS.

The question we need to ask ourselves is therefore, whether presence of a virus in semen equates with sexual transmission. Fortunately, in Medicine, when you think of a query, somebody else has considered it before and tried to answer it. The paper below from the United Kingdom- "Breadth of Viruses in Human Semen"- did just that in 2017, in the wake of the Zika virus outbreak. Apart from SARS, they documented 26 other viruses that appear in the human semen. Of these, less than half have ever had known sexual transmission. The list includes viruses you would never think of being passed on sexually, such as Chicken pox, Mumps and Chikungunya.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652425/

If one is observant, one will notice some glaring omissions in that list. Where's HPV, for example, that well known scourge of teenagers, the purveyor of genital warts, vulval and cervical cancer? Well, it's transmitted sexually, just not through semen. Presence in semen therefore does not equate with sexual transmission. Nor does the absence of a virus in semen provide reassurance that it is not transmitted sexually.

NEJM even ran an editorial on this in 2018- "Virus in Semen and the Risk of Sexual Transmission"-and I quote, "Contrary to prevalent belief, the detection of viral genomes in semen tends to be more common among viruses that are typically not sexually transmitted, such as certain adenoviruses, bunyaviruses, flaviviruses, hepadnaviruses, herpesviruses, paramyxoviruses, and retroviruses"

https://www.nejm.org/doi/full/10.1056/NEJMe1803212

There is another important issue- these COVID-19 virus particles found in semen may not be replication competent. RT-PCR only signifies the presence of the bit of the viral RNA that PCR probe primes with. It will do so regardless of whether the virus has a capsid or not- ie "live" or "dead".

Why Do Subjects of Afro-Caribbean Ancestry Have a Higher Mortality from COVID 19 than Caucasians?

The figures are startling. The mortality rate from Covid 19 among black Americans is 2.6 times that of Caucasians.

https://www.apmresearchlab.org/covid/deaths-by-race

In the UK, the figures are even more stark. Blacks are more than 4 times likely to die from COVID than whites.

https://www.ons.gov.uk/peoplepopulationandcommunity/birthsdeathsandmarriages/deaths/articles/coronavirusrelateddeathsbyethnicgroupenglandandwales/2march2020to10april2020

Although in general, Asians and Hispanics do worse than Caucasians as well, the differences are far less pronounced.

What accounts for this difference? There are various possibilities including genetic differences, acquired co-morbidities (diseases) or perhaps the way those co-morbidities are managed by physicians. The last one interests me the most.

Mendelian randomisation is nature's way of demonstrating differences in outcome due to a putative risk factor. For example, subjects with familial hypercholestrolaemia, the commonest autosomal dominant condition in the population, have a higher risk of heart disease and stroke than those without the condition, due to the fact that they have high LDL cholesterol. So far, no such signals have emerged in COVID.

However, differences could be acquired. Black subjects have a higher prevalence of hypertension and obesity than other races. Both of these have emerged as significant risk factors for COVID related mortality.

While the above is undoubtedly true, I believe (I haven't seen this in the medical press yet) that there is another factor- how hypertension is managed in Black subjects. For unknown reasons, Black people with hypertension respond poorly to a class of drugs called ACE inhibitors (yes, it's the same ACE you read about in the context of COVID receptors). In fact, there is evidence to suggest that Black subjects have a higher risk of death from MI (heart attack), stroke and heart failure when treated with ACE inhibitors than when not.

https://www.thecardiologyadvisor.com/home/topics/hypertension/ace-inhibitors-may-not-be-as-effective-in-black-patients/

As a result, ACE inhibitors are used far less often to treat hypertension in Blacks than in other races, and herein, I believe, lies the rub. I have cautioned here in the past against discontinuing ACE inhibitors in hypertensive subjects during the pandemic, as this is likely to lead to harm, an inference that was later confirmed by NEJM.

https://www.nejm.org/doi/full/10.1056/NEJMsr2005760 (free to access)

This is a case of unintended iatrogenic (physician induced) randomisation. If you are obese, and hypertensive, you are more likely to die from COVID 19. However, if you are obese, hypertensive and not taking ACE inhibitors, as in the majority of Black subjects, that risk is far higher.