Shelves and mentors

1 September 2015

Palese laboratoryWhen I became Peter Palese’s first Ph.D. student in 1976, his laboratory at Mt. Sinai School of Medicine in New York City was in dire need of shelves. The laboratory benches (pictured) had no room for storing the many bottles of reagents that I was beginning to generate.

When I told Peter that his laboratory needed shelves, he told me to get them. I looked in the yellow pages (there was no internet at the time) and found a dealer in downtown Manhattan. When I told Peter, he responded, to my amazement, that he would drive me there to get them.

Within minutes we were in Peter’s car, driving far downtown to a small garage filled with shelf parts. The proprietor loaded some into Peter’s car and we returned to Mt. Sinai, where we carried the metal pieces up the elevator into the laboratory. There I bolted them together and made one set of shelving for the low lab bench, and a second set for a higher bench. The photo of Peter shows what the shelves looked like just a few years ago.Peter Palese

A few days ago Matt Evans, a professor in the same department as Peter, told me that the Palese lab was being renovated and that the shelves that I had built were being discarded. He knew that I had made the shelves because I talk about them when I give a seminar. Matt was kind enough to send photos of the shelves, and of the low lab bench on which they once rested. It’s amazing that the shelves lasted for 39 years!

There is a reason why I’m writing this story, and why I tell it whenever I give a seminar. I had selected Peter to be my Ph.D. mentor, which meant that I listened to him. When he told me to build the shelves, I built them. When I went to David Baltimore’s laboratory as a postdoctoral fellow, I listened to David. When either Peter or David asked me to do something, I did it. I selected them as mentors because they had more experience in virology than I had and I wanted to train with them. Therefore when they asked me to jump, I said, ‘how high’?

Palese shelvesI am not saying that mentors should have the ability to make their trainees do whatever they ask. Of course it is important to question; if you don’t understand why your mentor is asking you to do a specific experiment, by all means ask them! Being an effective trainee means listening and questioning. But remember that in the end they have more experience doing science than you have. If you don’t want to listen, why did you pick them in the first place?

I have been very lucky to have trained many fine virologists during my 33 years at Columbia University. Most have listened to my suggestions. The best have always asked questions, leading to many wonderful two-way interactions. But some chose to consistently ignore my suggestions for experiments or projects. I have seen similar conduct in other laboratories here and elsewhere. This behavior baffles me. To this day, if Peter or David asked me to do something, I would immediately comply. They will always be my mentors.

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On episode #352 of the science show This Week in Virology, Vincent meets up with Michele Banks in Washington, DC to discuss her career as a creator of science-themed art.

You can find TWiV #352 at www.twiv.tv.

TWiV 351: The dengue code

23 August 2015

On episode #351 of the science show This Week in Virology, the Masters of the ScienTWIVic Universe discuss a novel poxvirus isolate from an immunosuppressed patient, H1N1 and the gain-of-function debate, and attenuation of dengue virus by recoding the genome.

You can find TWiV #351 at www.twiv.tv.

The individuals who believe that certain types of gain-of-function experiments should not be done because they are too dangerous (including Lipsitch, Osterholm, Wain-Hobson,) cite the 1977 influenza virus H1N1 strain as an example of a laboratory accident that has led to a global epidemic. A new analysis shows that the reappearance of the 1997 H1N1 virus has little relevance to the gain-of-function debate.

Human influenza viruses of the H3N2 subtype were circulating in May of 1977 when H1N1 viruses were identified in China and then Russia. These viruses spread globally and continue to circulate to this day. The results of serological tests and genetic analysis indicated that these viruses were very similar to viruses of the same subtype which circulated in 1950 (I was in the Palese laboratory in 1977 when these finding emerged). Three hypotheses were suggested to explain the re-emergence of the H1N1 virus: a laboratory accident, deliberate release, or a vaccine trial.

Rozo and Gronvall have re-examined the available evidence for the origin of the 1977 H1N1 virus. While there is ample documentation of the extensive work done during the 1970s in the Soviet Union on biological weapons, there is no evidence that Biopreparat had attempted to weaponize influenza virus. The release of the 1977 H1N1 virus from a biological weapons program is therefore considered unlikely.

It is more likely that the 1977 H1N1 virus was released during testing of influenza virus vaccines. Many such trials were ongoing in the USSR and China during the 1960s-70s. C.M. Chu, a Chinese virologist, told Peter Palese that the H1N1 strain was in fact used in challenge studies of thousands of military recruits, an event which could have initiated the outbreak.

The hypothesis that the 1977 H1N1 virus accidentally escaped from a research laboratory is formally possible, but there are even less data to support this contention. Shortly after this virus emerged, WHO discounted the possibility of a laboratory accident, based on investigations of Soviet and Chinese laboratories. Furthermore, the H1N1 virus was isolated at nearly the same time in three distant areas of China, making release from a single laboratory unlikely.

It is of interest that with the onset of the gain-of-function debate, which began in 2011 with the adaptation of influenza H5N1 virus to aerosol transmission among ferrets, the ‘laboratory accident’ scenario for the emergence of the 1977 strain has been increasingly used as an example of why certain types of experiments are ‘too dangerous’ to be done (See graph, upper left). For example, Wain-Hobson says that ‘1977 H1N1 represented an accidental reintroduction of an old vaccine strain pre-1957, probably from a Russian research lab’. Furmanski writes that ‘The virus may have escaped from a lab attempting to prepare an attenuated H1N1 vaccine’. In the debate on gain-of-function experiments, the laboratory escape hypothesis is prominently featured in public presentations.

The use of an unproven hypothesis to support the view that some research is too dangerous to do is another example of how those opposed to gain-of-function research bend the truth to advance their position. I have previously explained how Lipsitch incorrectly represented the results of the H5N1 ferret transmission studies. We should not be surprised at this tactic. After all, Lipsitch originally called for a debate on the gain-of-function issue, then shortly thereafter declared that the moratorium should be permanent.

Rozo and Gronvall conclude that the use of the 1977 influenza epidemic as a cautionary tale is wrong, because it is more likely that it was the result of a vaccine trial and not a single laboratory accident:

While the events that led to the 1977 influenza epidemic cannot preclude a future consequential accident stemming from the laboratory, it remains likely that to this date, there has been no real-world example of a laboratory accident that has led to a global epidemic.

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On episode #350 of the science show This Week in Virology, Vincent speaks with Katherine High about her career and her work on using viral gene therapy to treat inherited disorders.

This episode is drawn from one of twenty-six video interviews with leading scientists who have made significant contributions to the field of virology, part of the new edition of the textbook Principles of Virology.

You can find TWiV #350 at www.twiv.tv.

arenavirusI have a soft spot in my heart for Lassa virus: a non-fictional account of its discovery in Africa in 1969 inspired me to become a virologist. Hence papers on this virus always catch my attention, such as one describing its origin and evolution.

Lassa virus, a member of the Arenavirus family, is very different from Ebolavirus (a filovirus), but both are zoonotic pathogens that may cause hemorrhagic fever. It is responsible for tens of thousands of hospitalizations, and thousands of deaths each year, mainly in Sierra Leone, Guinea, Liberia, and Nigeria. Most human Lassa virus outbreaks are caused by multiple exposures to urine or feces from the multimammate mouse, Mastomys natalensis, which is the reservoir of the virus in nature. In contrast, outbreaks of Ebolavirus infection typically originate with a crossover from an animal reservoir, followed by human to human transmission. Despite being studied for nearly 50 years, until recently the nucleotide sequences of only 12 Lassa virus genomes had been determined.

To remedy this lack of Lassa virus genome information, the authors collected clinical samples from patients in Sierra Leone and Nigeria between 2008 and 2013. From these and other sources they determined the sequences of 183 Lassa virus genomes from humans, 11 viral genomes from M. natalensis, and two viral genomes from laboratory stocks. All the data are publicly available at NCBI. Analysis of the data lead to the following conclusions:

  • Lassa virus forms four clades, three in Nigeria and one in Sierra Leona/Liberia (members of a clade evolved from a common ancestor).
  • Most Lassa virus infections are a consequence of multiple, independent transmissions from the rodent reservoir.
  • Modern-day Lassa virus  strains probably originated at least 1,000 years ago in Nigeria, then spread to Sierra Leone as recently as 150 years ago. The lineage is most likely much older, but how much cannot be calculated from the data.
  • The genetic diversity of Lassa virus in individual hosts is an order of magnitude greater than the diversity of Ebolavirus. Furthermore, Lassa virus diversity in the rodent host is greater than in humans, likely a consequence of the longer, persistent infections that take place in the mouse.
  • The gene encoding the Lassa virus glycoprotein is subject to high selection in hosts, leading to variants that interfere with antibody binding.
  • Genetic variants that arise in one rodent are not transmitted to another.

Perhaps the most important result from this work is the establishment of laboratories in Sierra Leone and Nigeria that can safely collect and process samples from patients infected with Lassa virus, a BSL-4 pathogen.

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PoliovirusesIn midsummer 1986, five years after starting my poliovirus laboratory at Columbia University, I received a letter from Frederick L. Schaffer, a virologist at the University of California, Berkeley, asking if I would like to have his collection of poliovirus stocks. He was retiring and the samples needed a home, otherwise they would be destroyed. Of course I jumped at the opportunity to have a bit of virology history.

Three boxes full of dry ice arrived in the laboratory in August 1986. In them were sixty-six containers of different polioviruses that Dr. Schaffer had collected over the years. All three poliovirus serotypes were represented, both wild type and vaccine strains. The tubes were marked with dates ranging from 1948 to 1965. Most had come into Dr. Schaffer’s hands from well known poliovirologists, including Jonas Salk, Albert Sabin, Igor Tamm, Renato Dulbecco, and Charles Armstrong.

All of the samples were in glass containers, either tubes with a screw cap or a rubber stopper held in place with tape (pictured). A few were in glass bottles of the type that were used to grow cells. The collection held not a single plastic tube: these were the days before plastics entered the virology world. All were identified by hand-written labels on white cloth tape. Some of the labels in the photo read: Polio 1 Brunhilde (1963), Polio 2 MEF1, Polio 2 P712, HeLa P3, 10-21-62. Nearly all the samples were virus-containing cell culture supernatants.

Lansing poliovirusPerhaps the most amazing sample was a specimen labeled ‘Lansing poliomyelitis virus, 9/27/50, passage 379, C. Armstrong, NIH’. Within the glass vial was a intact mouse brain still attached to the spinal cord (there were no cell phones in 1986, hence no photo). The Lansing type 2 strain of poliovirus had been adapted to grow in mice by Charles Armstrong, and this was apparently the 379th intracerebral mouse-to-mouse passage! It was especially exciting for me to receive this sample, because my laboratory had been studying the ability of the Lansing strain of poliovirus to infect mice. I believe that the note accompanying the tube is in Dr. Armstrong’s writing.

I have since transferred most of the samples to plastic tubes which are stored in a -70C freezer. There is a trove of information to be obtained by studying these samples, but there are few poliovirologists left who are interested. Once poliomyelitis is eradicated – perhaps within the next 10 years – these samples, and similar ones throughout the world, will have to be destroyed.

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On episode #349 of the science show This Week in Virology, Vincent, Alan and Rich explain how to make a functional ribosome with tethered subunits, and review the results of a phase III VSV-vectored Ebolavirus vaccine trial in Guinea.

You can find TWiV #349 at www.twiv.tv.

filovirionAn Ebolavirus vaccine has shown promising results in a clinical trial in Guinea. This vaccine has been in development since 2004 and was made possible by advances in basic virology of the past 40 years.

The ability to produce the Ebolavirus vaccine, called rVSV-EBOV, originates in the 1970s with the discovery of the enzyme reverse transcriptase, the development of recombinant DNA technology, and the ability to rapidly and accurately determine the sequence of nucleic acids. These advances came together in 1981 when it was shown that cloned DNA copies of RNA viral genomes (a bacteriophage, a retrovirus, and poliovirus), carried in a bacterial plasmid, were infectious when introduced into mammalian cells. Production of an infectious DNA copy of the genome of vesicular stomatitis virus (VSV) was reported in 1995. In their paper the authors noted:

Because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.

This technology was subsequently used in 2004 to produce replication competent VSV carrying the genes encoding the glycoproteins of filoviruses, which others had shown are the targets of neutralizing antibodies. When injected into mice, these recombinant viruses induced neutralizing antibodies that were protective against lethal disease after challenge with Ebolavirus.

In a series of experiments done over the next 10 years, rVSV-EBOV was shown to protect nonhuman primates from lethal disease. In these experiments, animals were injected intramuscularly with the vaccine and challenged with Ebolavirus. The vaccine induced protection against lethal disease and prevented viremia. Extensive studies of the VSV vector in ~80 nonhuman primates showed no serious side effects, and only transient vector viremia.

The rVSV-EBOV was originally developed by Public Health Agency of Canada, and subsequently licensed to NewLink Genetics. Financial support has been provided from Canadian and US governments and others. From 2005 to the present, the NIH Rocky Mountain Laboratory in Hamilton, Montana has also been involved in this work, particularly with nohuman primate challenge studies. In November 2014 Merck entered an agreement with NewLink to manufacture and distribute the vaccine.

In August 2014, well into West Africa Ebolavirus outbreak, Canada donated 800 vials of vaccine to WHO, which then established the VSV Ebola Consortium (VEBCON) to conduct human trials.

The results of Phase I trials of rVSV-EBOV in Africa (Gabon, Kenya) and Europe (Hamburg, Geneva) were published on 1 April 2015. These trials comprised three open-label, dose-escalation trials, and one randomized, double blind controlled trial in 158 adults. Each volunteer was given one injection of 300,000 to 50 million plaque-forming units of rVSV-EBOV or placebo. No serious vaccine related events were reported, but immunization was accompanied by fever, joint pain, and some vesicular dermatitis. A transient systemic infection was observed, followed by development of Ebolavirus-specific antibody responses in all participants, and neutralizing antibodies in most.

The interim results of a phase III trial of rVSV-EBOV, begun on 23 March 2015 in Guinea, have just been published. It is a cluster-randomized trial with a novel design that is modeled on the ring vaccination approach used for smallpox eradication in the 1970s. In ring vaccination, individuals in the area of an outbreak are immunized, in contrast to treating a larger segment of the population. During this trial, when a case of Ebolavirus infection was identified, all contacts and contacts-of-contacts were identified. Some of these individuals were immediately immunized intramuscularly with 2 x 107 PFU, and others (randomly chosen) were immunized three weeks later. The primary outcome was Ebolavirus disease confirmed by PCR. As new cases arose in other areas (clusters), these were treated in the same way, hence the name of cluster-randomized trial.

The press has widely reported that the vaccine was ‘100% protective’. This outcome sounds much better than is represented by the data, so let’s look at the numbers.

Zero cases of Ebolavirus disease were observed in 2,014 immediately vaccinated people, while 16 cases were identified in those given delayed vaccine (n=2,380). These numbers were used to calculate the vaccine efficacy of 100%. While statistically significant, the numbers are small.

More telling are the results obtained when we consider all individuals eligible for immunization, not just those who were immunized (some were excluded for a variety of reasons). Of 4,123 eligible individuals, 2,014 were immunized as noted above, but 2,109 did not receive vaccine. Eight cases of Ebola virus disease were noted in the non-immunized population. This number is small, a consequence of the fact that the outbreak is waning.

On the basis of these interim results, the data and safety monitoring board decided that the trial should continue. However because the board felt that the vaccine is a success, they decided to curtail randomization of subjects into immediately vaccinated and delayed vaccinated groups. Now all contacts and contacts-of-contacts will immediately receive vaccine. As a consequence of this change, it will not be possible to improve the accuracy of vaccine efficacy. For example, when many more individuals are immunized in the future, many fewer that 100% might be protected from disease.

There are two lessons I would like you to remember from this brief history of an Ebolavirus vaccine. Developing a vaccine takes a long time (minimum 11 years for rVSV-EBOV) and depends on advances made with both basic and clinical research.  Don’t believe anyone who says that this vaccine was made in a year. And always look at the numbers when you hear that a vaccine has 100% efficacy.

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TWiV 348: Chicken shift

2 August 2015

On episode #348 of the science show This Week in Virology, Vincent and Rich discuss fruit fly viruses, one year without polio in Nigeria, and a permissive Marek’s disease viral vaccine that allows transmission of virulent viruses.

You can find TWiV #348 at www.twiv.tv.