Why Do Genes and Mutations Matter in SARS-CoV-2?

Why should we concern ourselves with genetics and mutations in SARS-CoV-2? There are many reasons why these are of interest, including the rate at which mutations are occurring. One reason is that mutations can result in immune evasion. Changes can occur in recognition sites (epitopes) for antibodies and for cytotoxic T cells, and these could, in theory, make vaccines less effective. In fact, our immune response tends to select for exactly these kinds of mutational changes in the virus. Another reason for our interest is that mutational signatures allow us to identify chains of transmission and origins of clusters of related mutants. Comparisons of genome sequences between current and previously circulating strains  may allow us to trace the current pandemic back to a common ancestral virus and, perhaps, identify its natural source, making it easier to anticipate and better deal with the next “spillover” event. Mutations may allow a virus to become more or less virulent or transmissible. Knowledge of the sequence of viral genes can lead to the prediction of the 3D structure of viral proteins, such as the RNA-dependent RNA polymerase and viral proteases, and inform the development of effective antiviral drugs. Sequence analysis will also allow us to characterize drug resistant mutants and develop alternative drugs. Let’s consider some of these points in a little more detail.

First, though, RNA viruses generally have a high mutation rate. Some mutations have no effect on the eventual protein sequence and are called silent or synonymous. Others alter the amino acid sequence of the protein and can result in a neutral effect or an increase or decrease in protein function. These are called non-synonymous, and most have no effect on protein function. Their RNA-dependent RNA polymerases have a high intrinsic error rate. SARS-CoV-2 seems to have a mutation rate much lower than many RNA viruses. This is because it has a proofreading nuclease that removes misincorporated bases. This is both good and bad news. It’s good because it makes the virus less likely to generate escape mutants from the immune response or from antiviral drugs. It’s potentially bad news because it could cause difficulties for drugs that target the viral polymerase by causing misincorporation of modified nucleotides, such as remdesivir. Fortunately in spite of this, remdesivir causes chain termination of RNA synthesis in an in vitro SARS-CoV-2 RNA replication system three bases downstream of its site of incorporation (1), so perhaps it won’t be a problem.

Is the virus mutating to become more infectious or pathogenic? Early in the epidemic a claim was made that there were two genetically distinguishable forms of SARS-CoV-2, called the S and L forms, in which the S form was ancestral to the L form and that the L form was spreading more aggressively, based on a relative frequency of 30% for the S form and 70% for the L. This has been debunked as being reflective of limited sampling, skewing the results, as well as founder effects, in which a single infected traveler seeds a new community with a single viral genotype. Later analyses showed the S form again becoming more predominant. More recently, similar observations have been made using a larger number of samples and focusing on the gene encoding the spike protein. They suggested that a mutation in the spike protein is becoming more dominant, thus, increasing a genotype variation, but here the same criticism would apply. Interestingly, they also present evidence for recombinant forms of SARS-CoV-2, suggesting some people become infected with more than one genotype. Nothing in the data reports differences in pathogenicity among different genotypes.

What about the origins of the virus? This is unclear and may remain so. Not only does the high number of coronavirus species make finding the exact ancestor problematic, but the proclivity of coronaviruses to recombine with each other adds considerable difficulty. The possibility that it was genetically engineered seems unlikely (2), among other reasons, because it is not optimally engineered. Two possibilities remain: that it directly infected humans or that it first infected an intermediate species, was adapted, and then infected humans, perhaps in the Wuhan wet market. The closest known related bat virus is more than 96% identical by nucleotide sequence to SARS-CoV-2. While this sounds quite close, its spike protein diverges in the receptor binding domain, suggesting that it may not bind efficiently to the human receptor, ACE2.. The Wuhan Institute of Virology scientists had a continuing study in which they collected bats and their blood and feces from caves in Yunnan Province to identify and catalog coronaviruses in response to SARS-1, and, in anticipation of future pandemics. Could one of them have become infected by inadvertently inhaling bat guano? It is a short high-speed train ride to Wuhan. Or could local villagers have acquired the virus? Bat guano is used in traditional Chinese medicine and is sometimes put in the eyes. You can buy it on Amazon. The virus may have been rare in humans until recently. Usually, however, transmission occurs through an intermediate host; civet cats with SARS-1 and camels with MERS. A fascinating book on the subject is Spillover, by David Quammen. Although pangolins have been suggested as an intermediate host, there is no convincing evidence so far. Perhaps in this case, humans are serving as the intermediate hosts. The definitive answer will require further sequencing efforts or it may never be answered conclusively.  Genetic studies of the virus have shown worldwide geographic routes of transmission that have occurred; see https://nextstrain.org/ncov/global.

Genomic sequences that have specific mutations can be thought of as having a convenient barcode that allows chains of transmissions to be traced. This can be highly valuable to epidemiologists. For example, a man who had traveled from China and arrived in Washington state in January tested positive for SARS-CoV-2. His virus had three distinct mutations that identified Wuhan as its origin. More than a month later, a high school student tested positive. The virus had the same mutations plus some new mutations. This led to the conclusion that the virus had been circulating in the general population during that time interval. Infections on the Grand Princess had the same mutational signature. Similar studies showed that the epidemic in New York had its origins primarily in travelers from Europe. The epidemic in Europe was mostly seeded by viruses from China. California virus samples also showed similarity to viruses from China but were distinct from the Washington state cluster, indicating a separate introduction, probably a bit earlier than those in Washington.

Another useful aspect of genetic studies is that they provide tools to understand potential targets for the immune system and by doing so, facilitate the development of vaccines. As an example, a study looked at B and T cell epitopes identified for SARS-CoV1 infections, then researchers looked for genetically similar regions in SARS-CoV-2 to identify possible immune targets (3). The authors further noted that these parts of the genome, which encode structural proteins of the virus, show very little variation among a large number of isolates, suggesting they may represent regions where the virus does not have much mutational latitude. Another method uses nucleotide sequences to determine the epitope , then synthesizes an overlapping set of peptides representing subsets of the epitope. These synthesized peptides are then be tested against various antisera, including natural immune sera, to identify regions of the protein that are immune targets. The viral genes can be cloned and expressed as protective antigens or directly synthesized proteins can be used for vaccine development…. Nucleotide sequence analysis, along with recombinant DNA techniques, have become extremely efficient, powerful and inexpensive, and are one of the main reasons that our understanding of emerging viruses comes as rapidly as it now does.

  1. C. J. Gordon et al., Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency. J Biol Chem, (2020).
  2. K. G. Andersen, A. Rambaut, W. I. Lipkin, E. C. Holmes, R. F. Garry, The proximal origin of SARS-CoV-2. Nat Med 26, 450-452 (2020).
  3. S. F. Ahmed, A. A. Quadeer, M. R. McKay, Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies. Viruses 12, (2020).

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So Will and When Will We Have a Vaccine?

Just to cut to the chase, we just do not know; but we may hope for one year and a half to two years, maybe less. And that will be great, but we have to distinguish between getting early evidence of efficacy and getting a vaccine ready for mass vaccination. But let’s consider background material that suggests it is likely we will indeed have a vaccine.

First, it is helpful to consider different mechanisms of immunity. There are two general types of immunity, innate and adaptive. The innate system is quick and dirty and recognizes general patterns widely shared by pathogens, such as double stranded RNA or bacterial lipopolysaccharides, but not normally present. It is mediated through the activity of cytokines and involves inflammation. The other mechanism, the adaptive immune system, is slower to respond but more precise and is mediated through antibodies and cytotoxic T cells.

Second, there are several kinds of immunization. Transfusion of immune serum or of purified antibodies, called passive immunization, can protect temporarily against infection and disease. This is currently being tried in patients.  Another type of immunization may induce a general innate immune response. Administration of some vaccines appear to induce a temporary immunity against viruses other than their targets, probably through stimulation of innate immunity. In theory, they should be able to protect against a relatively broad array of targets, which might include SARS-CoV2. This is likely the basis for the activities of BCG, which is used as immunotherapy against bladder cancer, for example. Phase 3 trials are currently enrolling subjects in Australia (BRACE) and the Netherlands (BCG-CORONA). More recently, Dr. Konstantin Chumakov (Food and Drug Administration, Office of Vaccines Research and Review) and Dr. Robert Gallo (Institute of Human Virology, University of Maryland School of Medicine, Baltimore) of the Global Virus Network have suggested repurposing polio vaccine for use against SARS-CoV2 through its induction of innate immunity. Finally, targeted vaccines can stimulate both innate and adaptive immunity. Targeted vaccines are the most commonly used vaccines, as they are the most specific and can provide durable protection.

Is a targeted vaccine for SARS-CoV2 possible? Several lines of evidence suggest the answer is yes. Worry number one is whether immune responses to the virus will be protective. Sera from infected rhesus monkeys and infected people can neutralize the virus, indicating it is possible to generate an effective response given an appropriate stimulus.  A second concern is that the virus will mutate rapidly and escape the immunity induced by vaccines. However, available sequence data suggest the overall genetic variability might be low. The virus has an RNA proofreading mechanism that reduces its mutation rate. A hopeful finding is that immune sera from people infected with the SARS1 virus (which is only 79% related at the whole genome level) neutralizes the SARS2 virus, suggesting elicited immunity will be broadly reactive. Similarly, a monoclonal antibody has been reported that neutralizes both SARS-CoV1 and SARS-CoV2.

There are many antibody-based passive immunization approaches with human and animal serum and recovered antibodies in pre-clinical development. There are also currently more than 200 targeted vaccines in widely various stages of development, including at least seven phase 1 or 2 clinical trials, and there are currently at least eight different approaches to developing a vaccine. One approach is simply to inject one or more of the viral proteins. Two other approaches involve injection and cellular take-up of either RNA or DNA encoding one or more viral proteins. Two other approaches involve vaccination with a heterologous viral vector derived from adenovirus, lentivirus, measles, or influenza virus, which has been engineered to express SARS-CoV2 proteins. These viral vectors can be either replication incompetent or able to replicate to various extents. Another type of vaccine vehicle is a virus-like particle (VLP), made artificially from viral proteins but lacking a viral genome. The SARS-CoV2 protein of interest is attached to the outer surface of the VLP. The other two vaccine approaches use whole SARS-CoV2 virus, either in killed form or attenuated so that its ability to replicate is severely compromised.

Obviously, testing for safety and efficacy is critical. Animal testing provides a way to obtain preliminary safety and efficacy data from candidate vaccines relatively quickly, because animals can be challenged with live virus. Drawbacks are that they may not accurately mimic the pathogenesis in humans and their immune responses may differ from that of humans. A study from China reported that vaccination with inactivated virus protected 8 rhesus macaques from viral challenge, but the numbers are small. The vaccinated macaques also developed antibodies able to neutralize different strains of the virus. They observed no harmful effects, including enhancement, in which a vaccine induces an immune response that makes infection more severe.

The gold standard in vaccine efficacy testing is obviously controlled clinical trials in humans. There are two ways to do this. One is with a large cohort of people from a group that has a high attack rate from the virus. Obvious, the more susceptible the test population, the fewer the numbers and shorter the time period required for a definitive answer. The most straightforward way, however, is to challenge vaccinated and unvaccinated unpaid volunteers with virus. This has the advantage that far fewer numbers are needed than in trials using an unchallenged general population, and results are obtained more quickly. However, such an approach is obviously fraught with ethical concerns.

As mentioned above, there are at least seven phase 1 or 2 trials of targeted vaccines now underway, several of which hope to have a vaccine by fall. An RNA-based vaccine by Moderna (clinical trials ID NCT04283461) has been reviewed by the FDA for phase 2 testing, and it is hoped to start phase 3 trials this summer. A Chinese whole killed virus vaccine candidate by Sinopharm (Chinese Clinical Trial Registry IdentifierChiCTR2000031809) has reached phase 2 status. The Oxford Vaccine Group is conducting a phase 1/2 trial with a vaccine (clinical trials ID NCT04324606) based on a chimp adenoviral vaccine vector that reportedly has shown protection in rhesus macaques.  A vaccine developed by Pfizer and BioNTech, called BNT162, is based on modified RNA (clinical trial ID NCT04368728). A phase 1/2 trial is planned in Germany and the United States. Inovio’s vaccine, called INO4800, is a DNA-based vaccine. They are currently planning a phase 1 trial in the United States and a phase 1/2 trial in South Korea (clinical trials ID NCT04336410). CanSino Biologics has an adenovius 5-based vaccine candidate that the company announced has passed phase 1 trials and are initiating a phase 2 trial (clinical trials ID NCT04313127). Sinovac has initiated phase 1 trials with a vaccine candidate based on inactivated virus plus adjuvant (clinical trials ID NCT04352608).

With the large number of approaches, the relative genetic stability of the virus, and hopeful results from animal trials, it seems likely a vaccine will be successfully developed. The big question, of course, is how soon. The best case seems to be by fall of 2020. Pressure to speed up the schedule may spur further considerations of the ethics of human volunteer challenge studies. The development of better antiviral therapies may influence considerations of these types of trials, as it would make them somewhat safer.

 

Read the June 2, 2020 update here

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Testing for SARS-COV-2

Testing, Testing, Testing…

The uncertainties about SARS-CoV-2 include critical questions such as what the death rate is for infected people, and what the infection rate is for the general public. This makes testing for infection of critical importance. There are numerous ways to establish the presence of infection. One is recovery of live virus, which in case of SARS-CoV-2 requires culturing the virus in biosafety level 3 containment. Although this would seem to be a gold standard, BSL3 facilities are not widely available, so this type of test is not routinely done. Another is to test for viral proteins, known as antigen testing. This seems to be amenable to high throughput testing, but reliable diagnostic tests have not yet become available. A third is to detect and measure viral RNA, generally done by PCR amplification following reverse transcription of viral RNA. It can be performed relatively readily in high quantity, but it is only an indirect indication of live virus. Even badly fragmented RNA can register as a positive, and positive results simply indicate that virus is being shed from somewhere within a patient, or has been recently. Thus, although recovered patients and asymptomatic people can be viral RNA positive, it is unclear as to what extent they can actually transmit virus without confirming live virus by isolation in a BSL3 facility. Fourth, serologic tests for antibodies to the virus can be performed on a massive scale, but depend critically on what the false positive and false negative rates are for a given test, and these can vary considerably among different test kits. The presence of antibodies means a person has been or is infected but may not still be infected at the time of the test.  Fifth, PCR tests similar to viral RNA tests can detect spliced or subgenomic RNA. In infected cells, full length viral RNA is spliced into a variety of smaller RNAs in order to encode many of the viral proteins. Thus, the presence of spliced subgenomic RNA indicates the presence of currently infected cells somewhere within the test subject. This process is somewhat labor intensive.

  • Of than these types of tests, the two that are amenable to mass testing and currently being used are viral RNA analysis and serologic tests, which are being widely and increasingly employed. To date, by far most testing that has been reported is for viral RNA. RNA is usually quantified based upon how many cycles of polymerase chain reaction are required to generate a detectable signal. These types of tests are highly specific (defined as the rate of false positives), but may lack sensitivity (defined as the rate of false negatives. This is not because of an intrinsic lack of sensitivity, but is because detection depends on the site from which the tested sample is taken. While throat and nasal swabs give reasonable sensitivity, bronchial alveolar lavage would be more sensitive, but collection is probably too onerous to be practicable. Saliva has been suggested as being conveniently obtained, but provides somewhat less sensitivity than nasopharyngeal swabs.(1) Wolfel et al.(2) performed RNA analysis of a number of tissues from moderately sick infected individuals. Swabs, saliva and stool contained viral RNA, although no live virus could be obtained from stool samples. The swabs and saliva contained live virus. Live virus could not be obtained after 8 days, although viral RNA remained positive. Urine and serum samples were never positive for viral RNA. Thus, detection of RNA in a sample is not tantamount to detection of infectious virus in the sample, or even in the tested individual.

Serologic tests for antibodies are becoming widely available and have the potential to make widespread testing much faster. As mentioned above, the presence of antibodies can indicate either that a person is infected or has been infected, and so does not necessarily indicate the presence of live virus. Following infection, IgM is the first antibody class produced, and so is more of an indicator of recent infection than is IgG, which is produced later. IgG tends to persist longer than IgM, and more sensitive to detect than past infections. Some antibody tests detect both classes; others only one. Samples collected include drawn blood or finger pricks. Widespread use of these tests has been hampered somewhat by the large number of tests of varying quality that are available; it is currently somewhat of a Wild West situation.

The specificity and sensitivity of these tests, defined respectively as the rate of false positives and false negatives, is critical for their usefulness, depending on whether they’re used for mass screening or diagnostic purposes. For diagnostic testing, sensitivity and specificity are obviously both important. For mass testing, specificity is absolutely critical.  Let’s consider using a test that is 95% specific for a population of 100 people that is 5% infected. The test will detect the 5 infected people. In addition, ~5 uninfected people will test positive, leading to the conclusion that 10% of the population is infected, even though the true infection rate is only half that. For reference, the test made by Abbot, which will be used in mass testing, has been reported as 100% sensitive, 99.5% specific. The other test that is likely to be used in mass testing is one by Roche. As of this writing, the accuracy and sensitivity data are not available, but will presumably be similarly high. Consideration of specificity should be taken into account when evaluating reports of unexpectedly high seroprevalence such as the study from Santa Clara County. The article can be accessed here, along with a number of critiques. Mass testing in the future should lend a lot more clarity to the true situation. It would be of interest to follow up positive serologic tests with confirmatory tests for RNA.

 

  1. E. Williams, K. Bond, B. Zhang, M. Putland, D. A. Williamson, Saliva as a non-invasive specimen for detection of SARS-CoV-2. J Clin Microbiol, (2020).
  2. R. Wolfel et al., Virological assessment of hospitalized patients with COVID-2019. Nature, (2020).

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