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.

 

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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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Antivirals in the Time of Coronavirus

An Evaluation of Remdesivir and Hydroxychloroquine

Many years ago, efforts to contain HIV resulted in the first widespread successful use of antiviral drugs, including deoxynucleotide analogs such as AZT, able to terminate reverse transcription of viral RNA. The current coronavirus pandemic has spurred extensive efforts to identify effective and safe anti-SARS-CoV-2 drugs. Recent reports have excited interest in Gilead’s remdesivir. Remdesivir’s activity is somewhat analogous to AZT, in that it is a nucleotide analog, but it has a 3-OH group and is not therefore a classical RNA chain terminator. Nevertheless, an analysis in vitro shows that it causes termination of transcription of viral RNA by RNA-dependent RNA polymerase RDRP 3 residues after its incorporation. There are some excellent reports characterizing in vitro activity of remdesivir against SARS-CoV-2 RDRP (1-4).

Remdesivir use has some potential issues. It is not orally bioavailable and must be infused. Treatment duration is a question still to be answered. Cost could be an issue. The SARS-CoV-2 has a 5’-3’ exonuclease which potentially could remove inappropriate nucleotide analogs, but at least in vitro with purified RDRP, this does not appear to happen.

Remdesivir (credit: Ulrich Perrey / Pool via Reuters)

The big question is of course is what is its therapeutic value? The recent excitement has come from two reports. One, in the New England Journal of Medicine, reported results from an uncontrolled compassionate use trial(5). Of 53 patients, 68% showed clinical improvement. More dramatic results were reported during a faculty video discussion at University of Chicago. Out of 113 patients with severe disease treated with remdesivir, most were successfully discharged and only two died. Again, however, this amounts to an uncontrolled trial, although the results are quite encouraging. These results represent preliminary findings from a trial sponsored by Gilead with 2,400 severe disease cases and 1600 moderate disease cases which is comparing treatments of five days duration with those of 10 days duration, so there is no true control group. However, further results are eagerly anticipated.

It appears that at the very least, targeting RDRP will be a fruitful approach. The development of an orally bioavailable version of remdesivir seems important. Since the structure of the RDRP is known, design of more powerful inhibitors should be possible. Finally, processing of the viral polyproteins into their functional forms requires a viral protease, called 3CL, for which it should be possible to design inhibitors. Protease inhibitors have proven highly effective in the cases of HIV and hepatitis C virus and could prove a useful alternative or additional target against SARS-CoV-2. Candidates for 3CL inhibitor lead compounds have already been identified(6).

Hydroxychloroquine has also received some attention, although its benefits are not yet clear, and it has some risky cardiac side effects. Although hydroxychloroquine has some activity in vitro against SAR-CoV-2 as well as other coronaviruses, it does not appear to have anti-coronaviral activity in infected animals. There have been several small studies reported in Covid19 patients. One in France, published online, showed a reduction in viral RNA in patients treated with hydroxychloroquine ± azithromycin.  However, this was not a randomized trial, had small numbers of patients, and had a number of methodological flaws. The other study, a controlled multicenter trial in China with 75 patients treated and 75 in the control arm. No effect was noted on viral RNA levels, but some clinical benefit was observed. A trial in Brazil was halted due to cardiotoxocity. The effects of hydroxychloroquine have been attributed to alteration of the endosomal compartment, affecting viral entry; alteration of glycosylation of ACE2, the spike protein receptor, reducing affinity; and immunomodulation, lowering an overly active inflammatory response. Its usefulness in Covid19 thus remains unsettled.  More recently, a larger but still non-controlled study was reported by the Veterans Administration with 368 patients (hydroxychloroquine alone, n=97; hydroxychloroquine plus azithromycin, n=113; and no hydroxychloroquine, n=158).   No benefit was observed; deaths were higher in the hydroxychloroquine group. The benefit/risk ratio of hydroxychloroquine is still unclear. Novartis is initiating a large clinical trial in the US to determine efficacy that could help answer this question.

  1. Y. Gao et al., Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Science, (2020).
  2. 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).
  3. A. Shannon et al., Remdesivir and SARS-CoV-2: structural requirements at both nsp12 RdRp and nsp14 Exonuclease active-sites. Antiviral Res, 104793 (2020).
  4. T. P. Sheahan et al., An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice. Sci Transl Med, (2020).
  5. J. Grein et al., Compassionate Use of Remdesivir for Patients with Severe Covid-19. N Engl J Med, (2020).
  6. Z. Jin et al., Structure of M(pro) from COVID-19 virus and discovery of its inhibitors. Nature, (2020).

 

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