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• COVID vaccines were not tested in subjects such as MS and other autoimmune diseases
• MS patients treated with immunosuppressive therapies react poorly to the vaccines
• Standard prevention measures for COVID-19 are of paramount importance

The rapid development of COVID vaccines in a matter of months was a scientific tour de force; and the result of accumulated knowledge over a 20yr period of time. The vaccines were tested in healthy individuals given the pressure to produce vaccines quickly for the vast numbers of people who needed to be immunized. However, people with a variety of ailments were not included in the roll out clinical trials of the vaccines. In particular, testing was not done on the 3-5% of individuals with compromised immune systems such as autoimmune disease (MS on certain treatments in particular), various forms of cancer (particularly Lymphoma and Leukemia) and individuals with transplants or HIV.

The mRNA vaccines have an excellent safety profile and do not worsen autoimmune conditions such as MS.

Most patients with MS on different therapies do make antibodies in response the COVID vaccination. However, patients on Ocrevus, Kesimpta, Lemtrada and post bone marrow transplant make antibodies to new challenges poorly, including COVID vaccination. In our experience with Ocrevus we have not seen antibody production even after 3-4 booster shots. A recent study, however, reported that Ocrevus patients had cellular immune responses (there are two arms of the immune system – antibodies and cellular responses and both are protective). In other conditions a range of 15-80% of individuals do not make antibodies. Neither the makers of the vaccines nor the Federal Government have performed clinical trials of the vaccines in these at risk groups as yet. There is also no consensus about using booster vaccines to try to get a heightened immune response in at risk individuals; though there have been discussions about the possibility of testing this approach.

Not having antibodies against the Corona virus increases the risk for developing COVID-19 infection. If someone in these groups were unfortunate enough to get infected they can be treated with Remdesivir and Monoclonal Antibodies, which offer hope of preventing progression to more severe forms of the infection .

This leaves continued use of the preventive methods we have become accustomed to such as social distancing and crowd avoidance, masks and good ventilation. These precautions make a lot of sense for at risk individuals in spite of the CDC loosening of protective requirements for the general public.

THE INTEGRAL PART OF TREATMENTS FOR MS

Monoclonal antibody therapy has become an integral aspect of the armamentarium of treatments for MS. It is helpful to consider how we got to this point with these tools and how these antibodies work in MS in particular.

The 100^th monoclonal antibody (MAB) for treatment has been recently been approved by the FDA; so that it is fitting to look back on how we got to where we are over the 35yr since the first MAB was approved by the FDA in 1986.

Until 1975 polyclonal antibodies (a mixture of different antibodies) were the only tool available to use experimentally to react with a specific immune target on or in a cell (an antigen). This was a blunt edged sword because there could be unwanted cross-reactions with other targets which muddied the waters. At that time Cesar Milstein and George Kohler working in the UK developed a technique (the hybridoma technique) which produced monoclonal antibodies. Here they were able to immunize mice and then isolate individual B cells, the antibody producing cells, from the spleen. To prevent these cells from dying they fused them with a tumor cell line, which in fact immortalized the antibody-producing B cells. By testing each cell line produced from single hybrid B cells they could specifically identify the antibodies reacting with the target of interest. They coupled the antibodies with a fluorochrome (a chemical which fluoresces in a defined light wave length) so they could identify the cell bearing the antigen of interest by microscopy or by using a flow cytometer (this technique is still widely used today, including in our laboratory). The unconjugated antibody could be used for diverse purposes such as blocking an immune reaction or destroying a cell to which the antibody was found. This technique revolutionized Immunology and in recognition of this phenomenal technical advance as well as its potential as a clinical therapeutic tool Milstein and Kohler were awarded the Nobel prize in 1984.

Later the antibodies needed to be modified because mouse antibodies injected into humans produced severe reactions. It is helpful to understand the structure of an antibody molecule and its modifications to recognize how they can be used therapeutically.

The antibody molecule is made up of two pairs of protein molecules which results in a Y-like structure. The larger of the proteins (heavy chain) is coupled to a smaller protein (light chain). This is then coupled to an identical partner to produce the Y shape of the whole molecule. Most of the molecule is made up of a standard sequence of protein building blocks (amino acids) found in all antibodies of a particular type. The outer part of the heavy and light chains, however, is comprised of different amino acids in each different antibody (the hypervariable region). This alteration from a “normative” or standard amino acid sequence is the result of gene mixing in individual B cells. This process occurs to tailor make an antibody to best fit the antigen to which it binds.

The basic structure can also be modified to make a monoclonal antibody for therapeutic use.

The starting point is the antibody molecule from the mouse (murine). The outer ends of the heavy and light chains can be substituted with the same part of the heavy and light chains from a human antibody. This reduces the chance of an immune reaction when used therapeutically. One can then get more focused and just substitute the hypervariable regions from a human antibody. And finally, a human cell making a defined antibody can be used instead of the original mouse cell. This markedly reduces the adverse reactions to the therapeutic antibody. The kind of antibody is denoted by different endings of the name used (see above).

All sorts of variations on this theme produce an array of different antibody molecules. For example, a drug or toxin can be connected to the antibody which will then deliver the drug or toxin to the defined target. Two halves from different antibodies can be combined so for example, two different cells can be brought together. And finally modified portions of the antibody molecule can be used.

Fig. 2 | Antibody formats. Antibody formats include canonical (part a), antibody–drug conjugates (part b), bispecifics (part c) and fragments (part d). Fragments include antigen-binding fragments (Fabs), single-chain variable region (scFv) constructs and domain antibodies. Radiolabelled antibodies and antibody–immunotoxins are not shown. These formats can be further subcategorized, and antibodies can span classifications. There are at least 30 different bispecific formats, for example, some of which include fragments. Modified from Nature Reviews Drug Discovery.

The figure below demonstrates the exponential growth of monoclonal therapies which have been FDA approved. In addition, there are literally hundreds of monoclonal antibodies at different stages of development.

Only 8 of the 100 monoclonal antibodies are approved for neurological disease though this number is likely to increase.

In the past, most of the monoclonal antibodies used to treat MS were repurposed from their original intended use. Since we do not know the driver mechanisms in the immune system in MS the selection of monoclonal antibodies for use in MS is a hit or miss affair. This can lead to unintended consequences in some cases.

Alemtuzumab was originally used in oncology. It is directed against a ubiquitous molecule (labelled CD52) on the surface of immune cells. The effect of using this monoclonal antibody is to break up cells which are bound by the antibody. This depletes the immune system fully. When the immune system re-constitutes there is a diminution of immune cells which react with myelin, delaying MS inflammation. Ultimately re-treatment is needed in most to keep the state of depletion. After treatment, B cells reconstitute faster than T cells, and this can lead to antibody-mediated autoimmune diseases apart from MS.

Natalizumab is an example of a monoclonal antibody which works in a model for MS, Experimental Allergic Encephalomyelitis (EAE). This can be induced in a variety of animals. The monoclonal antibody binds alpha-4 integrin, a molecule on the surface of activated T cells. The effectiveness in EAE  predicted correctly, that it would work in MS by blocking immune cells from sticking to the inner surface of blood vessels; the initial stage for immune cells to enter the nervous system. This prevents their transit into the brain, interrupting the cycle of inflammation in MS. Unfortunately, the use of Natalizumab is complicated by the occurrence of a JC viral infection (Progressive Multifocal Leukoencephalopathy).

MS has been considered an autoimmune disease caused by disordered T cells. It was surprising to find that the elimination of B cells (targeting CD20 a molecule on the surface of B cells) had therapeutic benefit for MS. However, elimination of B cells leads to their re-constitution; a process which induces the development of both regulatory T and B cells; which is probably the mechanism by which the immune system is controlled in MS. The original molecule in this class is Rituximab, a chimeric molecule. It was initially used to treat lymphoma and leukemia. It lacked efficacy in the treatment of Lupus and had an increase of infections in Rheumatoid Arthritis. It was then tested in MS. A humanized monoclonal anti-CD20 antibody was subsequently used (Ocrelizumab) with significant benefit in reducing relapses, MRI change and disease progression. A fully humanized antibody has now been developed (Ofatumumab) with similar success in MS. One downside of these antibodies is that killing immune cells in the circulation stimulates cytokine release (these are protein inflammatory molecules) which causes infusion reactions.

Unfortunately, a number of monoclonal antibodies have been unsuccessful in development as MS therapies. In part this is due to the fact that their mechanism of action is not relevant to, or harmful to immune processes in MS.

Daclizumab is a humanized monoclonal antibody developed for cancer therapy. It binds to one part of the Interleukin 2 receptor (Interleukin 2 is a growth stimulating cytokine) which is expressed in activated T cells. The bound cells are then removed. The mechanism by which this antibody acts in MS is not clear. One possibility is that it stimulates development of NK (natural killer) cells which have regulatory effects. The monoclonal antibody reduced relapses and had MRI benefit but a severe autoimmune encephalitis developed in some patients and it was withdrawn from the market.

Thus, monoclonal antibodies have come to play a very important role in medicine in general, and in MS therapy in particular. Knowledge of these antibodies and their actions should help making more informed treatment decisions.

FINDING THE ANSWER TO MS IS LIKE:

• Solving the mystery

DECODING MS:

• MS is an autoimmune disease which damages myelin and starts a chronic degenerative process in the brain and spinal cord.
• Systemic autoimmune diseases like Lupus affect the whole body. They reflect a widespread derangement in immune function.
• Restricted autoimmune diseases like MS are confined to one tissue or organ. This type of disease is primarily due to a defect in function of certain immune cells known as T regulatory cells.
• The failure of T regulatory cells is the result of a long developmental process which precedes the onset of clinical MS; and which involves genetic inheritance as well as environmental influences acting on these cells to ultimately disturb their normal function.
• T regulatory cells normally act to suppress the function of other immune cells called T effector cells, which prevents the development of autoimmunity. In the case of autoimmunity such as MS, the T regulatory cells may not only fail to function but may change to function like T effector cells which cause inflammation and tissue (myelin) damage.
• The key to understanding this process in MS is to define the stages of function and malfunction of T regulatory cells and the resultant dysfunction of unrestrained T effector cells. This is the goal of the research we do at MSRI.
• Flow cytometers and Cell sorters are invaluable pieces of laboratory equipment which allow us to dissect the processes in immune cells which lead to the development of MS. Understanding these processes will also allow us to rationally design more specific treatments for MS.

WHAT IS FLOW CYTOMETRY?

Flow cytometry is a technique used to study the surface and interior of immune or other cells. It has many innovative uses but we mainly use it at MSRI as a cell biology tool to study immune cells. This technique is integral to and has revolutionized much of contemporary immunology.

First, cells are prepared for the analysis. Antibodies against defined molecules on the surface of the interior of the cells are reacted with the cells.  Each specific antibody is labelled with a small molecule called a fluorochrome – and different antibodies are labelled with different fluorochromes to distinguish between them. A suspension of the cells is then passed in single file through a flow cell where it interacts with a laser beam. When the cell is exposed to a particular and narrow wavelength of light from a laser the relevant fluorochrome fluoresces at a defined wavelength and this fluorescence and its intensity are then read by the flow cytometer. Different lasers excite different fluorochromes, so that more than 20 molecules can potentially be studied simultaneously in different cell populations.

So, for example we can distinguish between a cell with a surface that expresses A B C and D, etc., and differentiate it from a cell that expresses A B F and G. Because we can also look at molecules inside a cell as well we can determine whether these cells are similar or different in internal molecules which may indicate similar or different cell functions.

The analysis occurs at speeds of up to 1000 cells/sec so a large number of cells can be analyzed in a very short time. The results thus obtained can then be evaluated further with a specialized computer program. Unfortunately the cells are not preserved after using this technique so we can sample what is happening in a population of immune cells in MS but we cannot use the analyzed cells again. This limits our ability to investigate the function of a cell population repeatedly.

WHY A CELL SORTER?

The flow cytometer essentially destroys the cells which have been analyzed so that their function as live cells cannot be investigated further. The cell sorter works on a similar principle to the flow cytometer but only surface molecules can be interrogated so as to preserve the cells. We can then react a population of cells with a panel of antibodies, each with its own specific fluorochrome. We pass the cells through the flow channel but now we program the sorter to separate cell populations with defined characteristics of interest. So we end up for example, with one test tube of A B C and D and another with A B F and G. We can then experiment on the sorted cell populations to evaluate functional differences between them. In the case of MS we can determine whether a specific immune cell population is functioning normally or not before or after manipulation of a defined condition. This can give us insights on what goes wrong in these cells in MS.

The technique can also be modified to put a single characterized cell in a single well on a plate so that we can analyze gene function and gene regulation in individual cells derived from a population of cells. This is a powerful and enormously informative technique which gives us granular detail about how each cell in a population is acting. It allows us to tie together both surface characteristics of a cell with how its genes are acting. This can also allow us to probe both the effects of genes as well as the environment on cells which may be pathogenic in MS.We currently do not have a cell sorter at MSRI. Having this piece of equipment is vitally important to allow us to probe the cause of MS as well as evaluate potential therapies. Any donation you can make, or your gift of $25, $50, $100 or more a month will make a difference to thousands of lives as this research progresses to solve the mystery of MS.