Immune Serum and the promise of Monoclonal Antibodies Against COVID19

According to an article in JAMA, immune serum from convalescent subjects has been used successfully against COVID19. This shouldn't come as a surprise. The technique was used for SARS and Ebola with success, and is still used in the treatment of Tetanus, and prophylaxis against Hepatitis B, Rabies and Chicken Pox following exposure.

While the preliminary results are encouraging, I'd caution against regarding it as some kind of cure though. Apart from the difficulties of using it widely (one patient- one recipient), there are several caveats.

Firstly, the antibodies in immune serum may provide protection against the virus temporarily, but will stymie the development of intrinsic antibodies or a T-cell response and therefore leave the subject vulnerable against re-infection if the pandemic continues.

Secondly, under certain conditions, infused antibodies in immune serum can lead to a phenomenon called Antibody Dependant Enhancement (ADE), which leads to increased virulence of the same virus or a related virus.

The immunoglobulin molecule (antibody)- IgG for all practical purposes, has two principal components- the Fab or antigen binding fragment and the Fc, which is the bit that binds to receptors on macrophages, neutrophils, T-cells and antigen presenting cells. The mechanism for ADE goes like this- the infused IgG binds to say, the spike protein in COVID through the Fab portion, and causes a conformational change in the receptor binding domain of the spike protein. It then binds to the host cell- say macrophages in the lungs, through the Fc portion, and actually facilitates the entry of the virus into the cell, thus increasing the cytolytic effect of the virus, before the host immunity has upgeared to deal with this new invader.

Something similar happened while trying to treat MERS cases in the past with immune serum. This increased the vulnerability of some serum recipients to infection with SARS, thought to be due to ADE. Later, a research group showed that a similar thing could happen with SARS itself, i.e immune SARS serum enhancing SARS severity.

Thirdly, serum carries the risk of transmitting other infections. While certain infective agents are screened for routinely- HIV, Hep B, Hep C, Syphilis universally, Malaria, Chaga's disease and HTLV in endemic areas and CMV for transplant recipients, it is impossible to screen for all known infective agents.

Fourth, foreign serum can lead to a Type III immune reaction called serum sickness. Yes, this was first demonstrated with horse serum, but can happen with human to human serum transfer at low levels.

So, what's the solution?

The really encouraging news to emerge from the successful use of immune serum lies in exploiting the promise of monoclonal antibodies. These are already in widespread use for autoimmune diseases- RA, Crohn's, Psoriasis, Lupus, etc, but are increasingly finding their way into infectious diseases.

They are easy to manufacture from scratch- take weeks to months from conception to readiness for use, rather than years. You simply extract the immune serum from a convalescent person, identify the memory B cell that makes the antibody that imparts immunity to your putative antigen by flow cytometry, clone the variable region of the immunoglobulin heavy and light chains and attach it to a Fc fragment. You can even use the Fab fragments themselves without a Fc portion, as with Certolizumab in RA.

This is not just theoretical. Palivizumab, a monoclonal antibody to the F protein of Respiratory Syncytial Virus, is used for prophylaxis in vulnerable infants against RSV induced bronchioloitis.

During the Zaire Ebolavirus outbreak in 2014-6, a combination of 3 mouse-human chimeric antibodies (like infliximab or rituximab) entered RCT. Unfortunately the latter was abandoned because the Ebola epidemic ended, and recruitment petered out (this is one of the biggest barriers to get drug companies to invest in such technology for infectious diseases).

A monoclonal antibody against Zika virus was waiting to enter clinical trial in pregnant women in Zika endemic areas. Again, the epidemic ended, and the enthusiasm waned.

The limiting factor is of course, cost. Monoclonal antibodies are expensive to mass produce and without the certainty of getting their money back, pharmaceutical companies have no incentive to change that.

But COVID is about to change everything. The cost to the world economy is likely to run into hundreds of billions of pounds, dwarfing the cost of production of such drugs. If COVID continues apace, the governments of the world, particularly the richer ones, will have no option but to pool their resources and incentivise the pharmaceutical sector to invest in this. After all, success is guaranteed, this is a mature and well tested technology, several companies have expertise in it, and there is an overwhelming moral imperative to save lives.

If not now, when?

Mortality Patterns With COVID19

The New England Journal of Medicine has just published its first report on a series of COVID19 patients in ICU care, treated in Seattle. Although it was a small series of 24 patients, there are some instructive & sobering facts.

Firstly, cough and SOB are the danger signs, being the presenting features in almost 90% each. Fever was present in only 50%. Could it be that these slightly elderly (mean age 64) with underlying co-morbidities (58% had diabetes, 21% had CKD), are unable to mount a pyrogenic response?

Secondly, the outcome was dire. Fifty percent died, 3 (12.5%) are still ventilated and may well die, only 5/24 were discharged home and 4/24 are in hospital, but off ventilation.

What's killing them? All patients had hypoxaemic respiratory failure, but 17/24 had shock. There was no evidence of cardiac failure on Echo, so this is septic shock, requiring vasopressors.

Hypoxia and shock are a deadly combination and mostly seen in bacterial infections such as Pneumococcus, Staph or with Gram negatives, but microbiological studies showed no superinfection in these patients, either with bacteria or common respiratory viruses. it's fair to assume therefore that the cause of shock was COVID 19 itself.

You'd think this is unusual for a viral infection and you'd be right. However, it is well documented with 3 viruses. The first is a strain of Influenza A- H3N2, which is in fact a component of the trivalent or quadrivalent Influenza vaccine. In years when H3N2 circulates, the mortality rate is 2.7 times the other strains included in the vaccine.

The second is avian influenza A- H5N1, which has a case fatality rate of 60%. The third is another avian Influenza A strain- H7N9- which has a fatality rate of 27%.

The reason these other viruses haven't run amok is because they cause mainly sporadic infections, presumably due to low infectivity, and are therefore easily isolated. Unfortunately COVID19 appears to share their capacity to cause hypoxaemia and shock, while spreading much more rapidly.

Tocilizumab in COVID19

Tocilizumab is a humanized monoclonal antibody to IL-6 widely used in Rheumatology. Indications include RA, GCA, & Takayasu's arteritis.

There are some reports of it being useful in COVID19. This is not surprising, as there are similar reports of its use with the original SARS virus.

Presumably, in some of these cases, Tocilizumab helps turn down the cytokine storm, characterised by high levels of IL-6. It is important to mention that IL-6 plays an important role in infections, where it is instrumental in priming B cells, among its other functions.

However, IL-6 can also damage normal tissues quite quickly, which might be innocent bystanders not germane to the ongoing inflammatory process.

Viruses can increase the production of IL-6 through the aforementioned TLRs. TLR3 and TLR9 (cognate receptors for dsRNA and DNA respectively) act through an adapter protein called MyD88. The latter leads to two divergent pathways. While one pathway downstream of MyD88 proceeds to activate kinases IRAK1 & IRAK4 and subsequently through IRF 5&7, turns on the class I interferon genes in the nucleus, the other pathway proceeds down IRAK4 & IRAK2 to activate nuclear factor kappa B, and thus increases the production of cytokines such as TNF alpha and IL-6.

Normally, the interferon producing pathway dominates the inflammatory pathway, and most subjects overcome their viral infections through the viricidal effect of interferons.

For reasons not best understood though, in some subjects, the inflammatory pathway dominates, thus quickly leading to tissue damage, and sometimes, death. It is for these subjects that Tocilizumab might be useful.

In a similar vein, it is worth mentioning here that the IL-6 receptor activates JAK1 and JAK2 (also TYK2), which in turn, switches on STAT3. It is not inconceivable therefore that JAK inhibitors such as Tofacitinib, Baricitinib and Upadicitinib would be useful in patients with cytokine storm.

One curious effect of Tocilizumab is that it makes CRP virtually useless as a marker of inflammation. Subjects on Tocilizumab markedly reduce the production of CRP by liver, and ongoing inflammation may proceed with a normal CRP.

Antimalarials in COVID19

There have been quite a few reports on the efficacy of antimalarials- namely chloroquine & hydroxychloroquine in COVID-19, mostly from China. Some countries, specifically the Indian council of Medical Research have now put out recommendations, suggesting doctors likely to be exposed to COVID19 use hydroxychloroquine for prophylaxis. The basis for such recommendations is unclear. There is very little to commend use of these medications in the treatment of COVID19 except anecdotal reports, let alone for prophylaxis.

Presumably, these recommendations are based on some studies that show in vitro efficacy of hydroxychloroquine against certain viruses. however, there are no RCTs that show in vivo efficacy.

It is instructive that hydroxychloroquine is used principally for the treatment of autoimmune diseases such as SLE, Rheumatoid, sarcoid, DLE, etc. The mechanism by which it works in these conditions is thought to be by raising the pH of endosomes, including lysosomes. The latter operates in a facultatively acidic pH. Two immune processes that occur in lysosomes would be of relevance here.

Firstly, antigen presenting cells (APC) such as dendritic cells digest antigens internalized by Fc gamma receptors present on the cell surface. The latter of course are designed to bind to the Fc portion of IgG, and thus gain access to the antigen which the Fab portion of IgG has bound in turn. These antigens are digested in the acid environment of lysosomes and taken up in the groove of HLA Class I & HLA Class II molecules, to be presented by the APC to CD8+, and CD4+ T cells respectively. The former have particular relevance to killing viruses directly by cytolytic action, while the latter stimulate B cells through CD40-CD40L interaction. Hydroxycholoroquine abrogates this action, which is advantageous in conditions such as Lupus, where the Fc gamma portion of IgG binds to many autoantigens that would have been cleared from circulation in otherwise normal subjects. The latter is one of the principal drivers of Lupus.

A similar acid pH is required for partial digestion and activation of Toll like receptors (TLR). The 3 main TLRs dealing with viruses are all intracellular- TLR3, which binds ssRNA, TLR7, which binds dsRNA, and TLR9, which binds DNA. These TLRs are produced in the ER, and then chaperoned to endosomes by a protein called Unc90 3b, there to be partially digested and activated, ready to receive their cognate RNA or DNA. Again, turning down this function is particularly useful for SLE.

There is no conceivable reason why this should be useful for viral infections though. Perhaps the most important APC relating to viral infections is the plasmacytoid dendritic cell, which produces copious amounts of type I & type III interferons on antigenic stimulation. These interferons are viricidal, and therefore, turning down these functions would not be beneficial for viral infections.

There is a suggestion however, of a differential effect. Apparently, hydroxychloroquine differentially inhibits the processing of weak antigens such as auto-antigens in lupus while sparing the response to strong antigens such as viruses. Even if this were the case, it would only provide reassurance that the drug would not increase the risk of viral infections while being used in SLE. It would not connote a viricidal effect per se.

A Coronavirus Perspective

I wanted to make some quick points about Coronavirus, mainly from a medical perspective. Firstly, nature is a great leveller. Self enforced or socially enforced isolation means less flights, less travelling on roads, less consumption and reduced human activity. That leaves its mark on the planet...for the better. There is far less NO2 over Italy being picked up from space. The virus has achieved what years of climate conferences could not. If you push the dice too far, at some point, nature will find a way of redressing the balance.

Worries about economic growth, recessions etc are not misplaced. However, ever increasing growth at the cost of killing the planet is self defeating. As is often said, shrouds don't have pockets. We survived 5 or 10 years ago when we were poorer, and had a lower GDP, didn't we?

Second, a lot of hope is being laid at the door of vaccines. A vaccine won't be ready in time to stem the epidemic. Even if it is lab ready, it takes a long time to be ready for the bench. Abbreviating Phase I trials to speed a vaccine through Phases II & III will not have good consequences, as Gerald Ford found out in 1976 when he fast-tracked a vaccine to the general population after a swine flu scare. People died or fell ill with the jab while the swine-flu outbreak never materialised. Ford lost the election.

Finally, there's been quite a bit of talk about strengthening one's immune system to "prepare" for the virus. This is attractive in theory but may not have legs to stand on. Pharmacologically, there are far more ways of suppressing the immune system than stimulating it. And the latter, when implemented, is targeted towards various cancers rather than infections, for eg IL-2 many years ago for renal cancer, CTLA-4 antibodies such as ipilimumab for melanoma, PD-1 and PD-L1 checkpoint inhibitors for NSCC of lung, etc, and CAR-T for acute B-cell lymphoblastic leukaemias.

Furthermore, the premise that a strong immunity saves you from a virus itself needs to be examined carefully. While COVID-19 is more likely to kill the elderly and infirm, it has spared children, whose immune system is not fully developed before 2 years of age (that's why you use conjugate vaccine in very young children- they can't form antibodies to capsular polysaccharides, unless you tag on a protein). Furthermore, among the deceased were some young people without pre-existing illnesses- like the doctor in Wuhan who raised the alarm. These young men & women died of an hyperactive immune response, manifested as the cytokine storm, typically occurring during the second week of illness, with death occurring around 18 days after falling ill. This is the same sort of response that kills AIDS patients carrying an opportunistic infection after being started on HAART-called IRIS. It's the immune system doing the killing here, not the virus. There are case reports of COVID-19 patients being treated with JAK inhibitors, for example, to turn down the cytokine storm. For relatively young people therefore, it is by no means straightforward.

To find an explanation for this, looking at the way the immune system deals with the virus might be instructive. Acute viral infections are dealt with principally by CD8 positive cytotoxic T cells and Natural Killer cells, which directly lyse virus infected cells with perforin and granzymes, and through Type I and Type III interferons, which are released in large quantities by plasmacytoid dendritic cells. While the interferon pathway is meant to overcome viruses, it can be a double edged sword.

To illustrate, single stranded RNA from viruses stimulates Toll-like receptor 7, which then leads to switching on of an adaptor protein called MyD88. MyD88 can lead to two completely different pathways. One pathway leads through kinases caled IRAK1 & IRAK4 to turn on Interferon regulatory factors-5&7 in the nucleus, leading to production of alpha & beta interferons, which in turn stimulate several interferon inducible genes to fight the virus. The other pathway though leads through IRAK4 & IRAK2 to turn on NF kappa B, and thus produces cytokines like TNF alpha and IL-6 which lead to a lot of damage- ie necrosis of innocent bystander tissues through the cytokine storm.

My suspicion is that, in a subset of patients, COVID-19 is preferentially activating this second pathway.